Background: Cell proliferation and polarization are important processes in the maturation of an epithelium during development and injury repair. These processes need to be tightly regulated to ensure an epithelium with a good structural integrity. Cystic Fibrosis (CF) is associated with a lack of structural integrity of the airway epithelium, suggesting a defect in polarization, and ultimately results in infections and respiratory failure. We have recently shown that CF airway epithelia are associated with overexpression and apical localization of fibronectin, phenomena usually linked with defects in polarization. However, the link between CF Transmembrane conductance Regulator (CFTR) and polarization defect is unclear.
Methods: We compared primary cultures of CF (F508del mutation) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked-out for CFTR by CRISPR-Cas9 (CFTR KO) to control counterparts, grown at an air-liquid interface (ALI) as a trigger for polarization through the inhibition of proliferation.
Results: RNA sequencing on HAECs and qPCR revealed that the Wnt and TGFβ signaling pathways were deregulated in CF airway epithelia, suggesting an imbalance between proliferation and polarization. Indeed, Ki67 analysis revealed a significant decrease in cell proliferation after 3 days of ALI in the CFTR KO cells (P <0.0001). Furthermore, as shown by immunoblotting and immunofluorescence experiments, the expression of the main signal transducer of the Wnt signaling pathway, β catenin, was significantly lower in CFTR KO cells at day 15 of ALI (P < 0.05). This decrease was caused by enhanced degradation of β catenin at D15 of ALI, (P < 0.05), as demonstrated by a cycloheximide chase assay. This mechanism is known to be induced by Wnt pathway inhibition. Indeed, DKK1, an inhibitor of the Wnt pathway, is over-secreted in CFTR KO cells. Moreover, an ectopic apical fibronectin deposition was observed in the CFTR KO cells during ALI, suggesting an impairment in polarization potentially due to aberrant TGFβ signaling. In an attempt to correct these differences, cells were treated with CHIR99021 and SB431542, Wnt pathway activator and TGFβ pathway inhibitor respectively. Treatment with CHIR99021 was able to restore the expression of β catenin in CFTR KO filters. Interestingly, apical and total fibronectin expression was also decreased after treatment. On the other hand, treatment with SB431542 led to a decrease in the phosphorylation of Smad2, one of the main signal transducers of the TGFβ pathway, as well as a decrease in total and apical fibronectin expression in CFTR KO cells.
Conclusions: Altogether, these results hint towards an interplay of the Wnt and TGFβ pathways in the proliferation and polarization of airway epithelial cells which are dysfunctional in the absence of CFTR. Further investigation is underway to provide more insight towards the initiation and progression of CF.
