SPLUNC1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration.

The multi-organ disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) that lead to diminished transepithelial anion transport. CF lungs are characterised by airway surface liquid (ASL) dehydration, chronic infection/inflammation and neutrophilia. Dysfunctional CFTR may upregulate the epithelial Na+ channel (ENaC), further exacerbating dehydration. We previously demonstrated that short palate lung and nasal epithelial clone 1 (SPLUNC1) negatively regulates ENaC in normal airway epithelia.Here, we used pulmonary tissue samples, sputum and human bronchial epithelial cells (HBECs) to determine whether SPLUNC1 could regulate ENaC in a CF-like environment.We found reduced endogenous SPLUNC1 in CF secretions, and rapid degradation of recombinant SPLUNC1 (rSPLUNC1) by CF secretions. Normal sputum, containing SPLUNC1 and SPLUNC1-derived peptides, inhibited ENaC in both normal and CF HBECs. Conversely, CF sputum activated ENaC, and rSPLUNC1 could not reverse this phenomenon. Additionally, we observed upregulation of ENaC protein levels in human CF bronchi. Unlike SPLUNC1, the novel SPLUNC1-derived peptide SPX-101 resisted protease degradation, bound apically to HBECs, inhibited ENaC and prevented ASL dehydration following extended pre-incubation with CF sputum.Our data indicate that CF mucosal secretions drive ASL hyperabsorption and that protease-resistant peptides, e.g. SPX-101, can reverse this effect to rehydrate CF ASL.

Webster MJ ，Reidel B ，Tan CD ，Ghosh A ，Alexis NE ，Donaldson SH ，Kesimer M ，Ribeiro CMP ，Tarran R ... -  《-》

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

Hobbs CA ，Blanchard MG ，Alijevic O ，Tan CD ，Kellenberger S ，Bencharit S ，Cao R ，Kesimer M ，Walton WG ，Henderson AG ，Redinbo MR ，Stutts MJ ，Tarran R ... -  《-》

Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways.

Garland AL ，Walton WG ，Coakley RD ，Tan CD ，Gilmore RC ，Hobbs CA ，Tripathy A ，Clunes LA ，Bencharit S ，Stutts MJ ，Betts L ，Redinbo MR ，Tarran R ... -  《-》

SPX-101 is stable in and retains function after exposure to cystic fibrosis sputum.

In healthy lungs, epithelial sodium channel (ENaC) is regulated by short, palate, lung, and nasal clone 1 (SPLUNC1). In cystic fibrosis (CF), ENaC is hyperactivated in part due to a loss of SPLUNC1 function. We have developed SPX-101 to replace the lost function of SPLUNC1 in the CF lung. Expression of SPLUNC1 was determined in sputum from healthy and CF donors. Stability of SPLUNC1, S18 (the ENaC regulatory domain of SPLUNC1), and SPX-101 was determined in sputum from CF donors and towards neutrophil elastase. Activity of SPX-101 after exposure to CF sputum was determined in airway epithelial cells from CF donors and in the βENaC transgenic mouse model. SPLUNC1 protein expression is significantly reduced in CF as compared to healthy sputum. SPLUNC1 is rapidly degraded in CF sputum as well as by a number of individual proteases known to be found in the sputum. SPX-101, but not S18, is stable in CF sputum. Finally, SPX-101 retains its ability to internalize ENaC, regulate airway surface liquid height, and increase survival of βENaC mice after exposure to CF sputum. Our results demonstrate that SPX-101, but not SPLUNC1 or S18, is stable in CF sputum. These results support the therapeutic development of SPX-101 for the treatment of cystic fibrosis.

Sesma JI ，Wu B ，Stuhlmiller TJ ，Scott DW ... -  《-》

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage.

Garcia-Caballero A ，Rasmussen JE ，Gaillard E ，Watson MJ ，Olsen JC ，Donaldson SH ，Stutts MJ ，Tarran R ... -  《-》