Chloride secretion by cultures of pig tracheal gland cells

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.     

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.     

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.     

Inhibition of mucin secretion with MARCKS-related peptide improves airway obstruction in a mouse model of asthma.

Allergic asthma is associated with airway epithelial cell mucous metaplasia and mucin hypersecretion, but the consequences of mucin hypersecretion on airway function are unclear. Recently, a peptide derived from the myristoylated alanine-rich C kinase substrate protein NH(2)-terminal sequence (MANS) was shown to inhibit methacholine (MCh)-induced mucin secretion from airway mucous cells by >90%. We studied the effect of intranasal pretreatment with this peptide on specific airway conductance (sGaw) during challenge with MCh in mice with allergen-induced mucous cell metaplasia. sGaw was noninvasively measured in spontaneously breathing restrained mice, using a double-chamber plethysmograph. Pretreatment with MANS peptide, but not a control peptide [random NH(2)-terminal sequence (RNS)], resulted in partial inhibition of the fall in sGaw induced by 60 mM MCh (mean +/- SE; baseline 1.15 +/- 0.06; MANS/MCh 0.82 +/- 0.05; RNS/MCh 0.55 +/- 0.05 cmH(2)O/s). The protective effect of MANS was also seen in mice challenged with allergen for 3 consecutive days to increase airway hyperresponsiveness, although the degree of protection was less (baseline 1.1 +/- 0.08; MANS/MCh, 0.65 +/- 0.06; RNS/MCh 0.47 +/- 0.03 cmH(2)O/s). Because routine sGaw measurement in mice includes nasal airways, the effectiveness of MANS was also confirmed in mice breathing through their mouths after nasal occlusion (baseline 0.92 +/- 0.05; MANS/MCh 0.83 +/- 0.06; RNS/MCh 0.61 +/- 0.03 cmH(2)O/s). In all instances, sGaw in the MANS-pretreated group was approximately 35% higher than in RNS-treated controls, and mucous obstruction accounted for approximately 50% of the MCh-induced fall in sGaw. In summary, mucin secretion has a significant role in airway obstruction in a mouse model of allergic asthma, and strategies to inhibit mucin secretion merit further investigation.     

Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.     

Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease 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 and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.     

The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression

Mucin expression was studied during proliferation and differentiation of the enterocyte-like Caco-2 and goblet cell-like LS174T cell lines. Caco-2 cells express mRNAs of MUC1, MUC3, MUC4 and MUC5A/C whereas MUC2 and MUC6 mRNAs are virtually absent. Furthermore, MUC3 mRNA is expressed in a differentiation dependent manner, as is the case for enterocytes. Concomitantly MUC3 protein precursor (550 kDa) was detected in Caco-2 cells. In LS174T cells mucin mRNAs of MUC1, MUC2 and MUC6 are constitutively expressed at high levels, whereas MUC3, MUC4 and MUC5A/C mRNAs are present at low levels. At the protein level LS174T cells express the goblet cell specific mucin protein precursors MUC2, MUC5A/C and MUC6 with apparent molecular masses of about 600 kDa, 470/500 kDa and 400 kDa respectively. MUC3 protein is not detectable. Furthermore, human gallbladder mucin protein (470 kDa precursor), of which the gene has not yet been identified, is expressed in LS174T cells. In addition, synthesis and secretion of the goblet cell specific mature MUC2, MUC5A/C and human gallbladder mucin was demonstrated in LS174T cells. It is concluded that Caco-2 and LS174T cell lines provide excellentin vitro models to elucidate the cell-type specific mechanisms responsible for mucin expression.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DMEM Dulbecco's modified Eagle's medium - EMEM Eagle's minimum essential medium - Endo-H endo--N-acetylglucosaminidase H - HGBM human gallbladder mucin - dpc days past confluence - PBS phosphate buffered saline     

Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop

Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor alpha). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.     

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.     

Signal transduction of mucous secretion by bronchial gland cells

Bronchial glands, which consist of mucous and serous cells, are abundant in human airways, playing a major role in the airway secretion. Cl(-) secretion is accompanied by water transport to the lumen in the acinar cells of bronchial glands. Agonists that increase [Ca(2+)]i induce the Cl(-) secretion in bronchial glands. Ca(2+) release from a IP(3)-sensitive Ca(2+) pool at the apical portion stimulates and opens Ca(2+)-sensitive Cl(-) channels at the apical membrane, producing Cl(-) secretion in bronchial glands. K(+) channels at the basolateral membranes are Ca(2+)-sensitive and activated by Ca(2+) release from a cADPribose-sensitive Ca(2+) pool, maintaining the Cl(-) secretion in bronchial glands. Further, cADP ribose in concert with IP(3) induce [Ca(2+)]i oscillation, inducing Cl(-) secretion in bronchial glands. Some tyrosine kinases are involved in the Cl(-) secretion in bronchial glands. Mucous and serous cells in bronchial glands take part in mucin secretion and the secretion of defensive substances (glycoconjugates), respectively. [Ca(2+)]i oscillations are shown to play a central role in the exocytosis of secretory granules in serous cells of bronchial glands. Other signal transductions of mucin and glycoconjugates in airway gland cells remain to be studied, although agonists which increase [cAMP]i are also well known to induce mucin and glycoconjugate secretion from airway glands.     

Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression

The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.     

Mucins and their O-Glycans from human bronchial epithelial cell cultures

A longstanding question in obstructive airway disease is whether observed changes in mucin composition and/or posttranslational glycosylation are due to genetic or to environmental factors. We tested whether the mucins secreted by second-passage primary human bronchial epithelial cell cultures derived from noncystic fibrosis (CF) or CF patients have intrinsically different specific mucin compositions, and whether these mucins are glycosylated differently. Both CF and non-CF cultures produced MUC5B, predominantly, as judged by quantitative agarose gel Western blots with mucin-specific antibodies: MUC5B was present at approximately 10-fold higher levels than MUC5AC, consistent with our previous mRNA studies (Bernacki SH, Nelson AL, Abdullah L, Sheehan JK, Harris A, William DC, and Randell SH. Am J Respir Cell Mol Biol 20: 595-604, 1999). O-linked oligosaccharides released from purified non-CF and CF mucins and studied by HPLC mass spectrometry had highly variable glycan structures, and there were no observable differences between the two groups. Hence, there were no differences in either the specific mucins or their O-glycans that correlated with the CF phenotype under the noninfected/noninflammatory conditions of cell culture. We conclude that the differences observed in the mucins sampled directly from patients are most likely due to environmental factors relating to infection and/or inflammation.     

Regulation of MUC5AC mucin secretion by depletion of AQP5 in SPC-A1 cells

Airway mucus is regulated by many inflammatory mediators such as ILs, TNF-alpha, EGF, PGF2alpha, LT, and so on. Recently, the relationship between membrane ion channel and mucus production has been under investigation. The present study aimed to examine whether AQP5 was involved in modulation of mucin expression and secretion in airway submucosal gland cells (SPC-A1). A recombinant plasmid (pShAQP5) containing small hairpin RNA expression cassette targeting AQP5 sequence was constructed. In pShAQP5 transiently transfected cells, ELISA showed MUC5AC synthesis and secretion were increased by 57.9% and 85.3%, respectively, on day 5 after pShAQP5 transfection. While in five stably transfected clones (shAQP5-G1, G2, G3, A2, and A5), the upregulated levels of MUC5AC mRNA were 118%, 165%, 65%, 123%, and 38%, respectively. The elevated levels of MUC5AC synthesis and secretion varied from 59-156% and 33-166%, respectively. This is the first reliable investigation of the regulation of MUC5AC mucin secretion by silencing AQP5. Further study of the regulatory mechanism between AQPs and mucins may provide new strategies for development of novel antihypersecretory drugs in airway diseases.     

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.     

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.     

Novel MUC1 splice variants contribute to mucin overexpression in CFTR-deficient mice

A cystic fibrosis (CF) mouse expressing the human mucin MUC1 transgene (CFM) reverted the CF/Muc1(-/-) phenotype (little mucus accumulated in the intestine) to that of CF mice expressing mouse Muc1, which exhibited increased mucus accumulation. Western blots and immunohistochemical analysis showed that the MUC1 protein was markedly increased in CFM mice in which it was both membrane bound and secreted into the intestinal lumen. Studies to determine the reason for increased levels of the extracellular domain of MUC1 mucin identified mRNA and protein of two novel splice variants and the previously described secreted MUC1 lacking the cytoplasmic tail (MUC1/SEC). Novel MUC1 splice variants, CT80 and CT58, were both transmembrane proteins with cytoplasmic tails different from the normal MUC1. The MUC1-CT80 and MUC1/SEC forms are found expressed mainly in the CFM mice intestines. Thus MUC1 expression is increased, and it appears that alternate cytoplasmic tails may change its role in signaling. MUC1 could be an important contributor to the CF intestinal phenotype.     

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.