CFTR antisense phosphorothioate oligodeoxynucleotides (S-ODNs) induce tracheo-bronchial mucin (TBM) mRNA expression in human airway mucosa

Mucus hypersecretion is a critical component of cystic fibrosis (CF) pathogenesis. The effects of dysfunction of the cystic fibrosis transmembrane regulator (CFTR) on mucin expression were examined using the tracheo-bronchial mucin (TBM) gene as an indicator. TBM mRNA expression was assessed in a human bronchial epithelial cell line (HBE1) and human nasal mucosal explants in vitro. Antisense phosphorothioate oligodeoxynucleotides (S-ODN) to TBM suppressed baseline expression of TBM mRNA in both systems, but had no effect on glyceraldehyde phosphate dehydrogenase mRNA (GAPDH) expression. Sense and missense (multiple scrambled control oligonucleotides) S-ODNs had no effect. 8Br-cAMP and PGE1 significantly elevated TBM mRNA expression. These increases were also specifically inhibited by the antisense S-ODNs. In order to induce a CF-like state, S-ODN to CFTR were added to explants. Antisense CFTR S-ODNs were anticipated to reduce the expression of cellular CFTR protein, and the level of CFTR function. Antisense, but not sense or missense, CFTR S-ODN significantly increased TBM mRNA expression. These data suggest that mucin hypersecretion in CF may be a direct consequence of CFTR dysfunction; the specific mechanism through which this effect is mediated is not known.     

Secreted and transmembrane mucins inhibit gene transfer with AAV4 more efficiently than AAV5

Adeno-associated virus (AAV) is a promising vector for gene transfer in cystic fibrosis. AAV4 and AAV5 both bind to the apical surface of differentiated human airway epithelia, but only AAV5 infects. Both AAV4 and AAV5 require 2,3-linked sialic acid for binding. However, AAV5 interacts with sialic acid on N-linked carbohydrates, whereas AAV4 interacts with sialic acid on O-linked carbohydrates. Because mucin is decorated with O-linked carbohydrates, we hypothesized that mucin binds AAV4 and inhibits gene transfer. To evaluate the effect of secreted mucin, we studied mucin binding and gene transfer to COS cells and the basolateral membrane of well differentiated human airway epithelia. AAV4 bound mucin more efficiently than AAV5, and mucin inhibited gene transfer with AAV4. Moreover, O-glycosidase-pretreated mucin did not block gene transfer with AAV4. Similar to secreted mucin, the transmembrane mucin MUC1 inhibited gene transfer with AAV4 but not AAV5. MUC1 inhibited AAV4 by blocking internalization of the virus. Thus, O-linked carbohydrates of mucin are potent inhibitors of AAV4. Furthermore, whereas mucin plays an important role in innate host defense, its activity is specific; some vectors or pathogens are more resistant to its effects.     

The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress

Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H₂O₂), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H₂O₂, compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.     

Evidence of gene conversion in genes encoding the Gal/GalNac lectin complex of Entamoeba

The human gut parasite Entamoeba histolytica, uses a lectin complex on its cell surface to bind to mucin and to ligands on the intestinal epithelia. Binding to mucin is necessary for colonisation and binding to intestinal epithelia for invasion, therefore blocking this binding may protect against amoebiasis. Acquired protective immunity raised against the lectin complex should create a selection pressure to change the amino acid sequence of lectin genes in order to avoid future detection. We present evidence that gene conversion has occurred in lineages leading to E. histolytica strain HM1:IMSS and E. dispar strain SAW760. This evolutionary mechanism generates diversity and could contribute to immune evasion by the parasites.     

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.     

Changes in expression of ion channels and aquaporins mRNA during differentiation in normal human nasal epithelial cells

Integrity of the airway epithelium is important for pulmonary defense mechanisms to infection. The lining of the airway contains a diverse population of cell types. Understanding about progenitor-progeny relationships during renewal of airway epithelium is important for elucidating mechanisms of injury repair or oncogenesis. Primary culture of airway epithelia is a good model for studying differentiation process of epithelial cells. Ion channels and aquaporins(AQPs) play a critical role on ion and fluid transport across airway epithelia. However, changes in their expression during differentiation of airway epithelial cells have not been reported yet. This study was undertaken to identify isoforms of aquaporins in cultured normal human nasal epithelial cells (NHNE) and effects of various culture conditions on expression of differentiation markers and channels. 1. Degenerative RT-PCR revealed that AQP3 and AQP4 are expressed in cultured NHNE cells. 2. Culture of NHNE cells on permeable filters induced expression of mucin, aquaporins and CFTR. 3. Retinoic acid induced morphological changes in NHNE cells and inhibited their proliferation. The treatment of retinoic acid induced expression of mucin and CFTR, whereas it inhibited expression of cornifin. The effect of retinoic acid was enhanced by culture of cells on permeable filters. 4. Dexamethasone induced ENaC expression in NHNE cells grown on permeable supports only, but did not affect expression of mucin, aquaporins and CFTR. These results indicate that cultured NHNE cells express aquaporins (AQP3 and 4), CFTR and ENaC, and culture of NHNE cells on permeable filters induces differentiation in to mucosecretory and surface epithelial cells, and that effects of retinoic acid and dexamethasone on gene expression are affected by culture conditions.     

Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development.

The Muc-1 mucin is found as a transmembrane protein in the apical surface of glandular epithelia. To provide insight into possible functions, we have assessed the timing of expression and the distribution of the Muc-1 protein during mouse embryogenesis using three different techniques: RT-PCR, northern blots and immunohistochemistry. Our results indicate that Muc-1 expression correlates with epithelial differentiation in stomach, pancreas, lung, trachea, kidney and salivary glands. Once started, Muc-1 synthesis continually increases with time, mainly due to epithelial area growth. Our data suggest that expression of the Muc-1 gene is under spatial and temporal control during organogenesis. Although Muc-1 is present in different organs, its expression is not induced systemically, but according to the particular onset of epithelial polarization and branching morphogenesis of each individual organ. It is of particular interest that Muc-1 protein can be detected lining the apical surfaces of the developing lumens when the epithelium of these organs is still undergoing folding and branching, and glandular activity has not yet started. We speculate that Muc-1 may participate in epithelial sheet differentiation/lumen formation during early development of the organs known to express it. This speculation is based on: (1) the detection of Muc-1 expression early during organogenesis, (2) the defined apical localization in different epithelia, (3) the decrease in cell-cell interactions when Muc-1 protein is highly expressed and (4) the possible interaction of its cytoplasmic tail with the actin cytoskeleton. However, it remains to be established using in vitro systems, whether the temporal and local expression of the Muc-1 gene coincident with the morphogenetic events described here is relevant for the process.