Proprotein convertase inhibition promotes ciliated cell differentiation - a potential mechanism for the inhibition of Notch1 signalling by decanoyl-RVKR-chloromethylketone

Abstract

Chronic repetitive rounds of injury and repair in the airway lead to airway remodelling, including ciliated cell loss and mucous cell hyperplasia. Airway remodelling is mediated by many growth and differentiation factors including Notch1, which are proteolytically processed by proprotein convertases (PCs). The present study evaluated a novel approach for controlling basal cell-type determination based on the inhibition of PCs. It was found that decanoyl-RVKR-chloromethylketone (CMK), a PC inhibitor, promotes ciliated cell differentiation and has no effect on the ciliary beat frequency in air-liquid interface (ALI) cultures of human nasal epithelial cells (HNECs). Comparative microarray analysis revealed that CMK considerably increases ciliogenesis-related gene expression. Use of cell-permeable and cell-impermeable PC inhibitors suggests that intracellular PCs regulate basal cell-type determination in ALI culture. Furthermore, CMK effect on ciliated cell differentiation was reversed by a Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). CMK inhibited the processing of Notch1, a key regulator of basal cell differentiation toward secretory cell lineages in the airway epithelium, and down-regulated the expression of Notch1 target genes together with furin, a PC. Specific lentiviral shRNA-mediated knockdown of furin resulted in reduced Notch1 processing and increased numbers of ciliated cells in HNECs. Moreover, CMK inhibited Notch1 processing and promoted regeneration and ciliogenesis of the mouse nasal respiratory epithelium after ZnSO4 injury. These observations suggest that PC inhibition promotes airway ciliated cell differentiation, possibly through suppression of furin-mediated Notch1 processing.