Gel-forming Mucin MUC2 Research Articles

The utility of the lineage specific immunohistochemical stains SATB2, CDX2, and villin, and the mucin glycoproteins MUC2, MUC5AC, and MUC6 to distinguish pulmonary invasive mucinous adenocarcinoma from metastatic colorectal carcinoma

ContextThe lungs are a common site of tumor metastasis. While morphology and immunophenotype can help differentiate primary from metastatic tumors, distinguishing pulmonary invasive mucinous adenocarcinoma (PIMA) from metastatic colorectal adenocarcinoma (CRC) may occasionally be challenging due to overlapping morphological and immunohistochemical features. Lineage-specific markers such as CDX2, TTF-1, and napsin A are helpful with pulmonary non-mucinous adenocarcinoma (PNMA), however they are non-specific and insensitive when applied to PIMA. SATB2 is a newer marker that distinguishes CRC from upper gastrointestinal and pancreaticobiliary tumors; its utility in distinguishing CRC from PIMA has not been fully elucidated. ObjectiveTo evaluate the performance of lineage-specific and mucin glycoprotein immunostains in distinguishing PIMA and CRC. DesignWe stained tissue microarrays comprising 34 PNMA, 31 PIMA, and 32 CRC with CK7, CK20, SATB2, CDX2, villin, TTF-1, napsin A, and gel-forming mucins MUC2, MUC5AC, and MUC6. ResultsPIMA showed significant (>50% of cells) expression of SATB2 (6%), CDX2 (6%), villin (74%), TTF-1 (13%), and napsin A (23%). However, significant CK7 expression was seen in nearly all PIMA (30/31) and none of the metastatic CRC. ConclusionOur results suggest that CK7 remains one of the most useful markers for distinguishing primary PIMA from metastatic CRC. Expression of the mucin glycoproteins MUC5AC and MUC6 and lack of expression of MUC2 favored a diagnosis of PIMA, but expression of these markers was too heterogeneous to be of clinical utility. To our knowledge this is the only study comparing the immunohistochemical profile of PIMA and metastatic CRC in lung metastasectomy specimens.

Tandem repeats structure of gel-forming mucin domains could be revealed by SMRT sequencing data

Mucins are large glycoproteins that cover and protect epithelial surface of the body. Mucin domains of gel-forming mucins are rich in proline, threonine, and serine that are heavily glycosylated. These domains show great complexity with tandem repeats, thus make it difficult to study the sequences. With the coming of single molecule real-time (SMRT) sequencing technologies, we manage to present sequence structure of mucin domains via SMRT long reads for gel-forming mucins MUC2, MUC5AC, MUC5B and MUC6. Our study shows that for different individuals, single nucleotide polymorphisms could be found in mucin domains of MUC2, MUC5AC, MUC5B and MUC6, while different number of tandem repeats could be found in mucin domains of MUC2 and MUC6. Furthermore, we get the sequence of MUC2, MUC5AC, and MUC5B mucin domain in a Chinese individual for each nucleotide at accuracy of possibly 99.98–99.99%, 99.93–99.99%, and 99.76–99.99%, respectively. We report a new method to obtain DNA sequence of gel-forming mucin domains. This method will provided new insights on getting the sequence for Tandem Repeat parts which locate in coding region. With the sequences we obtained through this method, we can give more information for people to study the sequences of gel-forming mucin domains.

Muc5ac Expression Protects the Colonic Barrier in Experimental Colitis.

The mucus gel layer (MGL) lining the colon is integral to exclusion of bacteria and maintaining intestinal homeostasis in health and disease. Some MGL defects allowing bacteria to directly contact the colonic surface are commonly observed in ulcerative colitis (UC). The major macromolecular component of the colonic MGL is the secreted gel-forming mucin MUC2, whose expression is essential for homeostasis in health. In UC, another gel-forming mucin, MUC5AC, is induced. In mice, Muc5ac is protective during intestinal helminth infection. Here we tested the expression and functional role of MUC5AC/Muc5ac in UC biopsies and murine colitis. We measured MUC5AC/Muc5ac expression in UC biopsies and in dextran sulfate sodium (DSS) colitis. We performed DSS colitis in mice deficient in Muc5ac (Muc5ac-/-) to model the potential functional role of Muc5ac in colitis. To assess MGL integrity, we quantified bacterial-epithelial interaction and translocation to mesenteric lymph nodes. Antibiotic treatment and 16S rRNA gene sequencing were performed to directly investigate the role of bacteria in murine colitis. Colonic MUC5AC/Muc5ac mRNA expression increased significantly in active UC and murine colitis. Muc5ac-/- mice experienced worsened injury and inflammation in DSS colitis compared with control mice. This result was associated with increased bacterial-epithelial contact and translocation to the mesenteric lymph nodes. However, no change in microbial abundance or community composition was noted. Antibiotic treatment normalized colitis severity in Muc5ac-/- mice to that of antibiotic-treated control mice. MUC5AC/Muc5ac induction in the acutely inflamed colon controls injury by reducing bacterial breach of the MGL.

Dynamic Interactions of a Conserved Enterotoxigenic Escherichia coli Adhesin with Intestinal Mucins Govern Epithelium Engagement and Toxin Delivery.

At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design.

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyer's patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103(+) type. In addition to the gel-forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

WS17.2 Hydrostatic pressure induced stretch activates CFTR in native pulmonary epithelium

The small intestinal mucus is of one type and mainly consists of the goblet cell secreted gel-forming mucin MUC2. Another type of mucins is the membranetethered mucins MUC3, MUC12 and MUC17 that are anchored in the apical plasma membrane of enterocytes. These mucins build the enterocyte glycocalyx and are thought to protect the brush border membrane. Secretion of the gel-forming mucin MUC2 is triggered by the cholinergic agonist carbachol. The same stimuli cause the enterocytes to relocalize the CFTR channel to the apical membrane to secrete chloride and bicarbonate necessary for proper mucin unfolding and hydration. Simultaneously, the NHE exchanger is relocated from the apical membrane to limit apical secretion of protons that should have neutralized bicarbonate. Interestingly, at the same time the transmembrane mucin MUC17 is also relocalized from the apical membrane into intracellular vesicles [1]. Both CFTR and MUC17 have C-terminal PDZ-binding motifs and both also bind to the PDZ protein PDZK1. This is an adapter family protein that controls proper localization of plasma membrane proteins by linking them to the cytoskeleton. By using an intestinal human cell line as model system, the interaction of PDZK1 with CFTR and MUC17 is analyzed by immunofluorescent co-staining and coimmunoprecipitation of native and mutant constructs. Phospho-specific analyses of the proteins are carried out to characterize interaction regulation due to phosphorylation.

Composition and functional role of the mucus layers in the intestine.

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria

We normally live in symbiosis with approximately 10(13) bacteria present in the colon. Among the several mechanisms maintaining the bacteria/host balance, there is limited understanding of the structure, function, and properties of intestinal mucus. We now demonstrate that the mouse colonic mucus consists of two layers extending 150 mum above the epithelial cells. Proteomics revealed that both of these layers have similar protein composition, with the large gel-forming mucin Muc2 as the major structural component. The inner layer is densely packed, firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer layer is movable, has an expanded volume due to proteolytic cleavages of the Muc2 mucin, and is colonized by bacteria. Muc2(-/-) mice have bacteria in direct contact with the epithelial cells and far down in the crypts, explaining the inflammation and cancer development observed in these animals. These findings show that the Muc2 mucin can build a mucus barrier that separates bacteria from the colon epithelia and suggest that defects in this mucus can cause colon inflammation.

Mucinous ovarian tumors associated with pseudomyxoma peritonei of adenomucinosis type: immunohistochemical evidence that they are secondary tumors

Pseudomyxoma peritonei (PMP) is a clinical condition initially thought to be related to ovarian mucinous tumors; however, immunohistochemistry and molecular biology techniques have convincingly made the link to appendiceal mucinous neoplasms, resulting in changes in histologic and clinical approaches. The objective of this study was to compare the immunohistochemical profile of ovarian tumors associated with PMP and intestinal mucinous ovarian neoplasms without PMP. The study was retrospective and included 28 intestinal ovarian mucinous tumors selected from the files of the Division of Surgical Pathology of the University of Sao Paulo Medical School, from 1996 to 2005. Seven cases were associated with PMP of disseminated peritoneal adenomucinosis-type and all presented borderline histology. Immunohistochemical staining for mucin genes products (MUC1, MUC2, MUC5AC, and MUC6), CK7, CK20, CA19.9, and CA125 were performed in tissue microarrays. Of note, we detected differences in the expression of MUC2 and CK20 between cases with and without PMP. Comparisons of borderline histology with that of benign/malignant tumors also revealed differences in MUC2 and CK20. Our results confirm that there is a distinct profile of intestinal ovarian tumors associated with pseudomyxoma, particularly with respect to the expression of the gel-forming mucin MUC2. The profile of borderline tumors, even in cases without PMP, was distinct from that of other primary mucinous tumors of the intestinal type, suggesting that borderline histology may represent a secondary tumor or a less aggressive variant of PMP. An appendiceal origin seems the most probable for this group of neoplasias.

Enzymatic cleavage as a processing step in the maturation of Muc4/sialomucin complex

Cleavage of Muc4/SMC precursor into two subunits is an essential processing step for maturation and occurs within a GD-PH sequence. Recent evidence indicates that cleavage of the precursor of gel-forming mucin MUC2 within the same tetrapeptide sequence occurs by a non-enzymatic, autocatalytic cleavage at low pH, and in cells in the late secretory pathway. Here we provide evidence that the cleavage step of Muc4/SMC processing occurs by a proteolytic mechanism. First, processing of Muc4/SMC precursor to ASGP-2 was inhibited in the presence of the mechanism-based serine protease inhibitor, Pefabloc SC, under conditions that did not block synthesis of other proteins. This inhibition led to an increased level of the precursor. Second, neutralization of the acidic environment of the late secretory pathway with NH4Cl did not inhibit cleavage of Muc4/SMC precursor. These results indicate that the two mucins can be processed by cleavage at the same peptide site by different mechanisms.

Characterization of mouse muc6 and evidence of conservation of the gel-forming mucin gene cluster between human and mouse

Using degenerate primers designed from conserved cysteine-rich domains of gel-forming mucins, we cloned two new mouse mucin cDNAs. Blast searching showed that they belong to the same new gene assigned to chromosome 7 band F5. This gene is clustered with the three secreted large gel-forming mucins Muc2, Muc5ac, and Muc5b in a region that exhibits synteny with human chromosome 11p15. Computer analysis and sequence alignments with mucin genes predict that the new gene is composed of 33 exons and spans 30 kb from the initiation ATG codon to the Stop codon. Sequence similarities, domain organization of the deduced peptide, and expression analysis allow us to conclude that this newly cloned mouse gene is Muc6, i.e., the mouse ortholog of human MUC6. Like those of their human homologs, the genomic order and arrangement of the four mucins within the cluster of mucin genes are conserved.

Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype

Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G −) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G − cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.