The membrane-bound mucins: From cell signalling to transcriptional regulation and expression in epithelial cancers

The membrane-bound mucins belong to an ever-increasing family of O-glycoproteins. Based on their structure and localization at the cell surface they are thought to play important biological roles in cell–cell and cell–matrix interactions, in cell signalling and in modulating biological properties of cancer cells. Among them, MUC1 and MUC4 mucins are best characterized. Their altered expression in cancer (overexpression in the respiratory, gastro-intestinal, urogenital and hepato-biliary tracts) indicates an important role for these membrane-bound mucins in tumour progression, metastasis, cancer cell resistance to chemotherapeutics drugs and as specific markers of epithelial cancer cells. Some mechanisms responsible for MUC1 and MUC4 role in tumour cell properties have been deciphered recently. However, much remains to be done in order to understand the molecular mechanisms and signalling pathways that control the expression of membrane-bound mucins during the different steps of tumour progression toward adenocarcinoma and evaluate their potential as prognostic/diagnostic markers and as therapeutic tools. In this review we focus on the molecular mechanisms and signalling pathways known to control the expression of membrane-bound mucins in cancer. We will discuss the mechanisms of regulation at the promoter level (including genetic and epigenetic modifications) that may be responsible for the mucin altered pattern of expression in epithelial cancers.     

Inhibition of Escherichia coli O157:H7 attachment by interactions between lactic acid bacteria and intestinal epithelial cells

The intestinal epithelial cell (IEC) layer of the intestinal tract makes direct contact with a number of microbiota communities, including bacteria known to have deleterious health effects. IECs possess innate protective strategies against pathogenic challenge, which primarily involve the formation of a physicochemical barrier. Intestinal tract mucins are principal components of the mucus layer on epithelial surfaces, and perform a protective function against microbial damage. However, little is currently known regarding the interactions between probiotics/pathogens and epithelial cell mucins. The principal objective of this study was to determine the effects of Lactobacillus on the upregulation of MUC2 mucin and the subsequent inhibition of E. coli O157:H7 attachment to epithelial cells. In the current study, the attachment of E. coli O157:H7 to HT-29 intestinal epithelial cells was inhibited significantly by L. acidophilus A4 and its cell extracts. It is also important to note that the expression of MUC2 mucin was increased as the result of the addition of L. acidophilus A4 cell extracts (10.0 mg/ml), which also induced a significant reduction in the degree to which E. coli O157:H7 attached to epithelial cells. In addition, the mRNA levels of IL-8, IL-1beta, and TNF-alpha in HT-29 cells were significantly induced by treatment with L. acidophilus A4 extracts. These results indicate that MUC2 mucin and cytokines are important regulatory factors in the immune systems of the gut, and that selected lactobacilli may be able to induce the upregulation of MUC2 mucin and specific cytokines, thereby inhibiting the attachment of E. coli O157:H7.     

Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: physiological distribution and pathological significance

Mucin secreted by mucosal epithelial cells plays a role in the protection of the mucosal surface and also is involved in pathological processes. So far, MUC1-4, 5AC, 5B, 6-8, 11-13 and 15-17 genes coding the backbone mucin core protein have been identified in humans. Their diverse physiological distribution and pathological alterations have been reported. Trefoil factor family (TFF) peptides are mucin-associated molecules co-expressed with MUC mucins and involved in the maintenance of mucosal barrier and the biological behavior of epithelial and carcinoma cells. Intrahepatic biliary system is a route linking the bile canaliculi and the extrahepatic bile duct for the excretion of bile synthesized by hepatocytes. Biliary epithelial cells line in the intrahepatic biliary system, secreting mucin and other molecules involved in the maintenance and regulation of the system. In this review, the latest information regarding properties, expression profiles and regulation of MUC mucins and TFF peptides in the intrahepatic biliary system is summarized. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases and intrahepatic cholangiocarcinoma.     

A deficient TLR2 signaling promotes airway mucin production in Mycoplasma pneumoniae-infected allergic mice

The original hygiene hypothesis suggests that early childhood respiratory infections preceding allergen exposure may decrease the prevalence of allergic diseases. We have recently demonstrated that Mycoplasma pneumoniae infection preceding allergen exposure reduced allergic responses in mice. However, the molecular mechanisms underlying the protective role of M. pneumoniae in allergic responses, particularly airway mucin production, remain unclear. Wild-type and Toll-like receptor 2 (TLR2)-deficient mice with a respiratory M. pneumoniae infection preceding allergen (ovalbumin) challenge were utilized to determine the regulatory role of TLR2-IFN-gamma signaling pathway in airway mucin expression. Furthermore, air-liquid interface cultures of mouse primary tracheal epithelial cells were performed to examine the effects of IFN-gamma on mucin expression. In wild-type mice, M. pneumoniae infection preceding allergen challenge significantly reduced airway mucins but increased IFN-gamma. In sharp contrast, in TLR2-deficient mice, M. pneumoniae preceding allergen challenge resulted in increased mucin protein without a noticeable change of IFN-gamma. In cultured mouse primary tracheal epithelial cells, IFN-gamma was shown to directly inhibit mucin expression in a dose-dependent manner. Our study demonstrates for the first time that a respiratory M. pneumoniae infection preceding allergen challenge reduces airway epithelial mucin expression in part through TLR2-IFN-gamma signaling pathway. A bacterial infection in asthmatic subjects with weakened TLR2-IFN-gamma signaling may result in an exaggerated airway mucin production.     

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.     

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.     

Effects of dexamethasone on Muc5ac mucin production by primary airway goblet cells

Mucus hypersecretion associated with airway inflammation is reduced by glucocorticoids. Two mechanisms of glucocorticoid-mediated inhibition of mucus production have been proposed, direct inhibition of mucus production by airway epithelial cells and indirectly through inhibition of proinflammatory mediators that stimulate mucus production. In this study, we examined the effect of dexamethasone (DEX) on mRNA expression and synthesis of MUC5AC by A549 human lung adenocarcinoma cells as well as Muc5ac and total high-molecular-weight (HMW) mucins by primary rat tracheal surface epithelial (RTSE) cells. Our results showed that in primary RTSE cells, DEX 1) dose dependently suppressed Muc5ac mRNA levels, but the levels of cellular Muc5ac protein and HMW mucins were unaffected; 2) did not affect constitutive or UTP-stimulated mucin secretion; 3) enhanced the translation of Muc5ac; and 4) increased the stability of intracellular Muc5ac protein by a mechanism other than the inhibition of the proteasomal degradation. In A549 cells, however, DEX suppressed both MUC5AC mRNA levels and MUC5AC protein secretion in a dose-dependent manner. We conclude that whereas DEX inhibits the levels of Muc5ac mRNA in primary RTSE cells, the levels of Muc5ac protein remain unchanged as a consequence of increases in both translation and protein stability. Interestingly, some of the effects of DEX were opposite in a cell line.     

Functional modulation of enterocytes by gram-positive and gram-negative microorganisms

Clinical studies have suggested that so-called probiotic bacteria may be effective as therapy in inflammatory bowel disease. However, the molecular mechanisms of their interaction with the intestinal surface remain undefined. The influence of whole probiotic bacteria [Escherichia coli Nissle 1917 (EcN); probiotic mixture VSL#3 (PM)], bacterial cell lysates, and conditioned media on transepithelial resistance (TER), IL-8 secretion, mucin gene expression, and tight junction proteins were determined in T84 and HT-29 intestinal epithelial cells (IEC). In addition, effects on pathogen (Salmonella dublin)-induced alterations were analyzed. EcN as well as debris and cell extracts induced IL-8 secretion from IEC, whereas no such effect was observed following incubation with the PM. The PM and soluble protein(s) released from the PM increased TER, prevented pathogen-induced decrease in TER, and were shown to stabilize tight junctions. The PM induced expression of mucins in IEC, and these organisms as well as EcN diminished S. dublin-induced cell death. Inhibition of MAPKs with PD-98059 or SB-203580 significantly decreased alterations in IL-8 synthesis and mucin expression and affected the regulation of TER. Probiotics and protein(s) released by these organisms may functionally modulate the intestinal epithelium of the host by different mechanisms, including the competition of whole organisms for contact with the epithelial surface as well as stabilization of the cytoskeleton and barrier function and the induction of mucin expression. Gram-negative and gram-positive organisms differ in the mechanisms activated, and a combination of organisms might be more effective than the application of a single strain.     

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.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

Human eosinophils induce mucin production in airway epithelial cells via epidermal growth factor receptor activation.

Eosinophil recruitment and mucus hypersecretion are characteristic of asthmatic airway inflammation, but eosinophils have not been shown to induce mucin production. Because an epidermal growth factor receptor (EGFR) cascade induces MUC5AC mucin in airways, and because EGFR is up-regulated in asthmatic airways, we examined the effect of eosinophils on MUC5AC mucin production in NCI-H292 cells (a human airway epithelial cell line that produces mucins). Eosinophils were isolated from the peripheral blood of allergic patients, and their effects on MUC5AC mucin gene and protein synthesis were assessed using in situ hybridization and ELISAs. When IL-3 plus GM-CSF or IL-3 plus IL-5 were added to eosinophils cultured with NCI-H292 cells, MUC5AC mucin production increased; eosinophils or cytokines alone had no effect. Eosinophil supernatant obtained by culturing eosinophils with IL-3 plus GM-CSF or IL-3 plus IL-5 also increased MUC5AC synthesis in NCI-H292 cells, an effect that was prevented by selective EGFR inhibitors (AG1478, BIBX1522). Supernatant of activated eosinophils induced EGFR phosphorylation in NCI-H292 cells. Supernatant of activated eosinophils contained increased concentrations of TGF-alpha protein (an EGFR ligand) and induced up-regulation of TGF-alpha expression and release in NCI-H292 cells. A blocking Ab to TGF-alpha reduced activated eosinophil-induced MUC5AC synthesis in NCI-H292 cells. These results show that activated eosinophils induce mucin synthesis in human airway epithelial cells via EGFR activation, and they implicate TGF-alpha produced by eosinophils and epithelial cells in the EGFR activation that results in mucin production in human airway epithelium.     

Tumor cell MUC1 and CD43 are glycosylated differently with sialyl-Lewis a and x epitopes and show variable interactions with E-selectin under physiological flow conditions

The mucins secreted from the colon carcinoma cell line COLO 205 have the MUC1 and CD43 (leukosialin) as core proteins, where both carry sialyl-Lewis a and MUC1 sialyl-Lewis x epitopes. The adhesion of E-selectin expressing CHO cells to the coated mucins was analyzed in a flow system revealing that the MUC1 mucin adhered better than the CD43 mucin. One reason could be their different glycosylation, a difference that was explored by analyzing the biosynthesis of MUC1 and CD43 in COLO 205 cells. Both the MUC1 and CD43 mucins became sialyl-Lewis a reactive, but after different times as revealed by pulse-chase studies. However, only MUC1 became sialyl-Lewis x reactive. These differences suggest that MUC1 and CD43 are synthesized in different compartments of the cell. It was also observed that the mucins from colon carcinoma patients had MUC1-type mucins that carried both sialyl-Lewis a and x epitopes and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. One could hypothesize that colon carcinoma derived MUC1 is decorated with potent E-selectin epitopes, and that this could be one of several reasons for the involvement of MUC1 in cancer development.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by siman virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines.The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of ³⁵SO₄ were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons.These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.     

The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues.

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.     

Mucin glycoproteins in neoplasia

Mucins are high molecular weight glycoproteins that are heavily glycosylated with many oligosaccharide side chains linked O-glycosidically to the protein backbone. With the recent application of molecular biological methods, the structures of apomucins and regulation of mucin genes are beginning to be understood. At least nine human mucin genes have been identified to date. Although a complete protein sequence is known for only three human mucins (MUC1, MUC2, and MUC7), common motifs have been identified in many mucins. The pattern of tissue and cell-specific expression of these mucin genes are emerging, suggesting a distinct role for each member of this diverse mucin gene family. In epithelial cancers, many of the phenotypic markers for pre-malignant and malignant cells have been found on the carbohydrate and peptide moieties of mucin glycoproteins. The expression of carbohydrate antigens appears to be due to modification of peripheral carbohydrate structures and the exposure of inner core region carbohydrates. The expression of some of the sialylated carbohydrate antigens appears to correlate with poor prognosis and increased metastatic potential in some cancers. The exposure of peptide backbone structures of mucin glycoproteins in malignancies appears to be due to abnormal glycosylation during biosynthesis. Dysregulation of tissue and cell-specific expression of mucin genes also occurs in epithelial cancers. At present, the role of mucin glycoproteins in various stages of epithelial cell carcinogenesis (including the preneoplastic state and metastasis), in cancer diagnosis and immunotherapy is under investigation.     

Cystic fibrosis: An inherited disease affecting mucin-producing organs

Our current understanding of cystic fibrosis (CF) has revealed that the biophysical properties of mucus play a considerable role in the pathogenesis of the disease in view of the fact that most mucus-producing organs are affected in CF patients. In this review, we discuss the potential causal relationship between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the production of mucus with abnormal biophysical properties in the intestine and lungs, highlighting what has been learned from cell cultures and animal models that mimic CF pathogenesis. A similar cascade of events, including mucus obstruction, infection and inflammation, is common to all epithelia affected by impaired surface hydration. Hence, the main structural components of mucus, namely the polymeric, gel-forming mucins, are critical to the onset of the disease. Defective CFTR leads to epithelial surface dehydration, altered pH/electrolyte composition and mucin concentration. Further, it can influence mucin transition from the intracellular to extracellular environment, potentially resulting in aberrant mucus gel formation. While defective HCO₃⁻ production has long been identified as a feature of CF, it has only recently been considered as a key player in the transition phase of mucins. We conclude by examining the influence of mucins on the biophysical properties of CF sputum and discuss existing and novel therapies aimed at removing mucus from the lungs.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Excess Secretion of Gel-Forming Mucins and Associated Innate Defense Proteins with Defective Mucin Un-Packaging Underpin Gallbladder Mucocele Formation in Dogs

Mucosal protection of the gallbladder is vital yet we know very little about the mechanisms involved. In domestic dogs, an emergent syndrome referred to as gallbladder mucocele formation is characterized by excessive secretion of abnormal mucus that results in obstruction and rupture of the gallbladder. The cause of gallbladder mucocele formation is unknown. In these first mechanistic studies of this disease, we investigated normal and mucocele-forming dog gallbladders to determine the source, identity, biophysical properties, and protein associates of the culprit mucins with aim to identify causes for abnormal mucus behavior. We established that mucocele formation involves an adoptive excess secretion of gel forming mucins with abnormal properties by the gallbladder epithelium. The mucus is characterized by a disproportionally significant increase in Muc5ac relative to Muc5b, defective mucin un-packaging, and mucin-interacting innate defense proteins that are capable of dramatically altering the physical and functional properties of mucus. These findings provide an explanation for abnormal mucus behavior and based on similarity to mucus observed in the airways of people with cystic fibrosis, suggest that abnormal mechanisms for maintenance of gallbladder epithelial hydration may be an instigating factor for mucocele formation in dogs.