Mechanistic analysis and significance of sphingomyelinase‐mediated decreases in transepithelial CFTR currents in nHBEs

Abstract

Abstract Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508‐CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib‐1, which also decreased CFTR function. The SMase‐mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5′ adenosine monophosphate‐activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non‐CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508‐CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid‐SMase protein than nHBEs. Lastly, we determined that airway‐like neutrophils, which are increased in the CF lung, secrete acid‐SMase. Identifying the mechanism of SMase‐mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid‐SMase from cell types relevant to CF.

Keywords

• AMPK
• cystic fibrosis transmembrane conductance regulator
• epithelial cells
• modulator therapy
• neutrophils
• sphingomyelinase