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. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd

Quantitative mass spectrometry for human melanocortin peptides in vitro and in vivo suggests prominent roles for β-MSH and desacetyl α-MSH in energy homeostasis

“…The mechanism for leptin elevating POMC-derived peptide concentrations could be due to its previously observed effects on inducing POMC transcription and translation [26] , [28] , its effects on promoting prohormone processing by increasing PC1/3 and PC2 expression [29] , or some combination of the two. Conversely, treatment of cultures with a broad-spectrum PC inhibitor that is likely to inhibit many PC enzymes at the concentration used [90] , [91] significantly reduced concentrations of POMC-derived peptides. Together, these findings are consistent with observations made in animals, suggesting that this human cellular model system may have predictive power for understanding human-specific aspects of POMC biology, such as the regulation of β-MSH processing and secretion.…”

Furin as a therapeutic target in cystic fibrosis airways disease

“…Indeed, this hypothesis was tested in a study using a broad-spectrum inhibitor of furin, decanoyl-RVKR-CMK, which demonstrated that treatment of human nasal epithelial cells with this inhibitor showed reduced Notch1 processing and enhanced ciliated cell differentiation. Knockdown of furin in these cells also caused a reduction in Notch1 processing and an increase in ciliated cell numbers, further demonstrating the involvement of furin in this process [ 83 ].…”

Recent advances in human respiratory epithelium models for drug discovery

“…CRISPR/Cas9 mediated depletion of FoxJ1 and subsequent multi-parametric analysis of HBEC ALI models, showed significant reduction of mucociliary differentiation into the ciliated cell phenotype without affecting barrier integrity (Rapiteanu et al, 2020). Efficacy testing of novel compounds, such as proprotein convertase inhibitors, acting via Notchdependant mechanisms have been shown to promote ciliagenesis in the 3D ALI models (Lee et al, 2017).…”