Abstract Rationale The cystic fibrosis transmembrane conductance regulator (CFTR) channel controls epithelial ion and fluid homeostasis and has been suggested to influence progenitor cell differentiation by modulating signaling pathways such as PTEN, Wnt, and Notch. Given the high expression of CFTR in a subset of alveolar type 2 (AT2) cells, we hypothesized that CFTR plays a critical role in alveolar epithelial repair following injury. We propose that, beyond its canonical role in ion and fluid transport, CFTR expression in AT2 cells facilitates their proliferation and differentiation into alveolar type 1 (AT1) cells by modulating key regenerative signaling pathways. Methods Primary AT2 organoids derived from wild-type (WT) and CFTR-G551D cystic fibrosis (CF) ferrets were exposed to bleomycin (BLM), with or without the CFTR potentiator VX-770, and their regenerative responses were compared. Gene expression was quantified by qRT-PCR and confirmed through immunostaining. For in vivo studies, AT2-specific Cftr knockout (SPCCreER; Cftrflx/flx) mice and their wild-type littermates were subjected to BLM injury, followed by histopathologic analysis and assessment of AGER/YAP1 expression analysis. Results WT ferret AT2 cell organoids formed regular cystic structures with large lumens, whereas CF AT2 cell organoids exhibited irregular structures with high cell density and small lumens, along with a basal cell-like transcriptional marker bias and impaired differentiation into AT1-like cells, as determined by qRT-PCR. Upon BLM exposure, wild-type organoids exhibited a dose-dependent decrease in viability (LC50 ≈ 3 µg/mL), which was significantly blunted by VX-770, raising the LC50 by approximately 40%. Notably, BLM treatment of wild-type organoids increased the proportion of SPChigh cells co-expressing AT2 transitional markers (KRT7 and KRT8) while decreasing AGER expression, consistent with early transcriptional activation during alveolar epithelial repair. PCA analysis of qRT-PCR data showed that BLM-treated wild-type organoids clustered closely with CF organoids, and that VX-770 treatment reversed this shift. Notably, BLM-injured WT mice exhibited prominent Krt5+ basal cell bronchiolization and strong nuclear YAP1 staining, indicating active remodeling. In contrast, SPCCreER;Cftrflx/flx (AT2 cell Cftr conditional knockout) mice developed diffuse parenchymal consolidation with reduced nuclear YAP1 expression, consistent with impaired regeneration and supporting the critical role of YAP signaling in alveolar epithelial regeneration. Conclusion This study demonstrates that pharmacological enhancement of CFTR function mitigates BLM-induced aberrant AT2 cell differentiation in ferret AT2 cell organoids in vitro and in mouse lungs in vivo. These findings suggest that CFTR is a key regulator of AT2 cell viability and regenerative capacity during alveolar repair, possibly through a YAP-dependent pathway. This abstract is funded by: none
Yang et al. (Fri,) studied this question.