Airway epithelial cell differentiation relies on deficient Hedgehog signalling in COPD

Abstract

Background: Hedgehog (HH) pathway is constantly under scrutiny in the context of organ development. Lung morphogenesis requires HH signalling which participates thereafter to the pulmonary homeostasis by regulating epithelial cell quiescence and repair. Since epithelial remodelling is a hallmark of Chronic Obstructive Pulmonary Disease (COPD), we investigated whether the main molecular actors of HH pathway participate to airway epithelial cell differentiation and we analysed their alterations in COPD patients. Methods: Sonic HH (Shh) secretion was assessed by ELISA in airway epithelial cell (AEC) air-liquid interface culture supernatants. HH pathway activation was evaluated by RT-qPCR, western blot and immunostaining. Inhibition of HH signalling was achieved upon Shh chelation during epithelial cell differentiation. HH pathway core components localization was investigated in lung tissues from non-COPD and COPD patients. Findings: We demonstrate that progenitors of AEC produced Shh responsible for the activation of HH signalling during the process of differentiation. Preventing the ligand-induced HH activation led to the establishment of a remodelled epithelium with increased number of basal cells and reduced ciliogenesis. Gli2 activating transcription factor was demonstrated as a key-element in the regulation of AEC differentiation. More importantly, Gli2 and Smo were lost in AEC from COPD patients. Interpretation: Our data suggest that HH pathway is crucial for airway epithelial cell differentiation and highlight its role in COPD-associated epithelial remodelling.

Sonic hedgehog signalling as a potential endobronchial biomarker in COPD

“…Our results are consistent with our previous observations showing that prevention of ligand-induced HH activation resulted in epithelial remodelling with an increased number of basal cells and decreased ciliogenesis [15], therefore mimicking the remodelling features of COPD observed in endobronchial samples in our study.…”

“…In concordance with our recent observations on FFPE tissues from lung resections [15], Ptch1, the receptor responsible for transduction of Shh signalling, was also found on non-differentiated and differentiated cells from isolated AEC and biopsies. Gli2 may be found in basal AEC nuclei, but can also be observed subciliary in ciliated cells, suggesting the presence of autocrine and paracrine signalling in the epithelium.…”

“…We first identified a marked decrease in Gli2 nuclear localization of basal cells and Gli2 protein levels in bronchial epithelium, highlighting a defect in HH signalling in COPD. The observed decrease of Gli2+ nuclear staining of cells obtained by bronchial brushing was consistent with our previous findings [15]. The apparent discrepancy with the staining observed on bronchial biopsies could be explained by the different sources of biological material.…”

Consecutive Hypoxia Decreases Expression of NOTCH3, HEY1, CC10, and FOXJ1 via NKX2-1 Downregulation and Intermittent Hypoxia-Reoxygenation Increases Expression of BMP4, NOTCH1, MKI67, OCT4, and MUC5AC via HIF1A Upregulation in Human Bronchial Epithelial Cells

“…Although both NOTCH1 and NOTCH3 have been implicated in regulating the balanced differentiation of airway basal cells between the secretory and ciliated cell fates (Tsao et al, 2009;Gomi et al, 2015), both NOTCH3 and HEY1 mRNA expression was significantly decreased in the small airway epithelium of COPD patients (Tilley et al, 2009), while increased expression levels of activated NOTCH1 and the effector HEY2 were observed in COPD airways and mutant murine lungs and were associated with decreased numbers of ciliated cells and increased goblet cell metaplasia and mucus overproduction (Guseh et al, 2009;Boucherat et al, 2012), suggesting that NOTCH1 and NOTCH3 may play distinct roles in regulating airway epithelial differentiation and proliferation. Previous studies have demonstrated that enhanced squamous metaplasia and goblet cell hyperplasia as well as reduced ciliogenesis are all correlated with increased NOTCH signaling and are implicated in the COPD airways (Pilette et al, 2001;Guseh et al, 2009;Boucherat et al, 2012;Barnes, 2015;Gohy et al, 2019;Belgacemi et al, 2020). It has also been shown recently that reduced ciliogenesis in COPD airways is associated with overexpressed TGF-β1 and deficient Sonic hedgehog (Shh)/Gli2/Smo signaling (Gohy et al, 2019;Belgacemi et al, 2020).…”

“…Previous studies have demonstrated that enhanced squamous metaplasia and goblet cell hyperplasia as well as reduced ciliogenesis are all correlated with increased NOTCH signaling and are implicated in the COPD airways (Pilette et al, 2001;Guseh et al, 2009;Boucherat et al, 2012;Barnes, 2015;Gohy et al, 2019;Belgacemi et al, 2020). It has also been shown recently that reduced ciliogenesis in COPD airways is associated with overexpressed TGF-β1 and deficient Sonic hedgehog (Shh)/Gli2/Smo signaling (Gohy et al, 2019;Belgacemi et al, 2020). In this study, we show for the first time that NOTCH1 and NOTCH3 expression levels in the COPD airway epithelium are differentially regulated and respectively correlated with overexpression of the goblet cell marker MUC5AC and deficiency of the ciliated cell marker FOXJ1 (Figures 5, 7, 8).…”