The ER stress transducer IRE1ß is required for airway epithelial mucin production.

Inflammation of human bronchial epithelia (HBE) activates the endoplasmic reticulum (ER) stress transducer inositol-requiring enzyme 1 (IRE1)α, resulting in IRE1α-mediated cytokine production. Previous studies demonstrated ubiquitous expression of IRE1α and gut-restricted expression of IRE1ß. We found that IRE1ß is also expressed in HBE, is absent in human alveolar cells, and is upregulated in cystic fibrosis and asthmatic HBE. Studies with Ire1ß(-/-) mice and Calu-3 airway epithelia exhibiting IRE1ß knockdown or overexpression revealed that IRE1ß is expressed in airway mucous cells, is functionally required for airway mucin production, and this function is specific for IRE1ß vs. IRE1α. IRE1ß-dependent mucin production is mediated, at least in part, by activation of the transcription factor X-box binding protein-1 (XBP-1) and the resulting XBP-1-dependent transcription of anterior gradient homolog 2, a gene implicated in airway and intestinal epithelial mucin production. These novel findings suggest that IRE1ß is a potential mucous cell-specific therapeutic target for airway diseases characterized by mucin overproduction.

Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal surfaces of the airways.

Mucus, with its burden of inspired particulates and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be "watery" and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate (KS), a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed KS-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190-1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, whereas the smaller adenoassociated virus penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.