VAMP8 is a vesicle SNARE that regulates mucin secretion in airway goblet cells.

Mucin secretion is an innate defence mechanism, which is noxiously upregulated in obstructive lung diseases (e.g. chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma). Mucin granule exocytosis is regulated by specific protein complexes, but the SNARE exocytotic core has not been defined in airway goblet cells. In this study, we identify VAMP8 as one of the SNAREs regulating mucin granule exocytosis. VAMP8 mRNA was present in human airway and lung epithelial cells, and deep-sequencing and expression analyses of airway epithelial cells revealed that VAMP8 transcripts were expressed at 10 times higher levels than other VAMP mRNAs. In human airway epithelial cell cultures and freshly excised tissues, VAMP8 immunolocalised mainly to goblet cell mucin granules. The function of VAMP8 in airway mucin secretion was tested by RNA interference techniques. Both VAMP8 short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) reduced mucin secretion induced by PAR agonists, neutrophil elastase and ATP in two airway epithelial cell culture models. Notably, basal (non-agonist elicited) mucin secretion was also reduced in these experiments. VAMP8 knockdown was also effective in decreasing mucin secretion in airway epithelial cell cultures with induced mucous metaplasia/mucin hypersecretion. Unlike VAMP8 silencing, knockdown of VAMP2 or VAMP3 did not affect mucin secretion. Importantly, in VAMP8 knock-out (KO) mice with IL-13-induced mucous metaplasia, mucin content in the bronchoalveolar lavage (BAL) and ATP-stimulated mucin secretion in the trachea were reduced compared to WT-matched littermates. Our data indicate that VAMP8 is an essential SNARE in airway mucin granule exocytosis. Reduction of VAMP8 activity/expression may provide a novel therapeutic target to ameliorate airway mucus obstruction in lung diseases.

Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial Na+ channel-overexpressing mice: role of TNF-{alpha} and IL-4R{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.

Overexpression of the epithelial Na(+) channel beta subunit (Scnn1b gene, betaENaC protein) in transgenic (Tg) mouse airways dehydrates mucosal surfaces, producing mucus obstruction, inflammation, and neonatal mortality. Airway inflammation includes macrophage activation, neutrophil and eosinophil recruitment, and elevated KC, TNF-alpha, and chitinase levels. These changes recapitulate aspects of complex human obstructive airway diseases, but their molecular mechanisms are poorly understood. We used genetic and pharmacologic approaches to identify pathways relevant to the development of Scnn1b-Tg mouse lung pathology. Genetic deletion of TNF-alpha or its receptor, TNFR1, had no measurable effect on the phenotype. Deletion of IL-4Ralpha abolished transient mucous secretory cell (MuSC) abundance and eosinophilia normally observed in neonatal wild-type mice. Similarly, IL-4Ralpha deficiency decreased MuSC and eosinophils in neonatal Scnn1b-Tg mice, which correlated with improved neonatal survival. However, chronic lung pathology in adult Scnn1b-Tg mice was not affected by IL-4Ralpha status. Prednisolone treatment ablated eosinophilia and MuSC in adult Scnn1b-Tg mice, but did not decrease mucus plugging or neutrophilia. These studies demonstrate that: 1) normal neonatal mouse airway development entails an IL-4Ralpha-dependent, transient abundance of MuSC and eosinophils; 2) absence of IL-4Ralpha improved neonatal survival of Scnn1b-Tg mice, likely reflecting decreased formation of asphyxiating mucus plugs; and 3) in Scnn1b-Tg mice, neutrophilia, mucus obstruction, and airspace enlargement are IL-4Ralpha- and TNF-alpha-independent, and only MuSC and eosinophilia are sensitive to glucocorticoids. Thus, manipulation of multiple pathways will likely be required to treat the complex pathogenesis caused by airway surface dehydration.

Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.

RATIONALE: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in beta-epithelial Na(+) channel (betaENaC)-overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors. OBJECTIVES: The aim of this study was to identify the initiating lesions and investigate the natural progression of lung disease caused by airway surface dehydration. METHODS: Lung morphology, gene expression, bronchoalveolar lavage, and lung mechanics were studied at different ages in betaENaC-overexpressing mice. MEASUREMENTS AND MAIN RESULTS: Mucus obstruction in betaENaC-overexpressing mice originated in the trachea in the first days of life and was associated with hypoxia, airway epithelial necrosis, and death. In surviving betaENaC-overexpressing mice, mucus obstruction extended into the lungs and was accompanied by goblet cell metaplasia, increased mucin expression, and airway inflammation with transient perinatal increases in tumor necrosis factor-alpha and macrophages, IL-13 and eosinophils, and persistent increases in keratinocyte-derived cytokine (KC), neutrophils, and chitinases in the lung. betaENaC-overexpressing mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. CONCLUSIONS: Our studies demonstrate that airway surface dehydration is sufficient to initiate persistent neutrophilic airway inflammation with chronic airways mucus obstruction and to cause transient eosinophilic airway inflammation and emphysema. These results suggest that deficient airway surface hydration may play a critical role in the pathogenesis of chronic obstructive pulmonary diseases of different etiologies and serve as a target for novel therapies.