Canonical Wnt/β-Catenin Signaling Modulates C/EBPα Abundance and Alveolar Epithelial Differentiation Marker Expression Under Hyperoxia: An In Vitro Model of Bronchopulmonary Dysplasia

Background: Bronchopulmonary dysplasia reflects an alveolar epithelium in which the differentiation program remains incomplete. CCAAT/enhancer-binding protein α (C/EBPα) is indispensable for alveolar type II (AT2) maturation, yet the mechanisms governing its abundance under oxidant stress remain unexamined. Because canonical Wnt/β-catenin signaling has been reported to reduce C/EBPα in other lineages, we asked whether β-catenin signaling modulates C/EBPα in hyperoxia-exposed alveolar epithelium and whether this is accompanied by changes in differentiation marker expression.Methods: Rat AT2-like RLE-6TN cells were exposed to 95% O2 for 48 h. Wnt output was manipulated bidirectionally with ICG-001 (10 µM) under hyperoxia and lithium chloride (20 mM) under normoxia, while β-catenin was silenced under hyperoxia and overexpressed under normoxia. The contribution of C/EBPα was examined by silencing it during Wnt inhibition and by restoring it under continued hyperoxia without manipulating the pathway. Pathway activity (active β-catenin, Axin2), C/EBPα, differentiation markers (pro-SP-C/Sftpc, AQP5/Aqp5) and apoptosis were assessed.Results: Hyperoxia increased active β-catenin and Axin2 and reduced C/EBPα, pro-SP-C and AQP5, with increased apoptosis (all p < 0.001). ICG-001 reduced Axin2 without altering active β-catenin and increased C/EBPα, pro-SP-C and AQP5, whereas pharmacological glycogen synthase kinase-3β (GSK-3β) inhibition with lithium chloride reproduced the hyperoxic pattern under normoxia. β-catenin silencing under hyperoxia increased C/EBPα, Sftpc and Aqp5, whereas β-catenin overexpression under normoxia reduced them (all p < 0.001). Silencing C/EBPα markedly attenuated the ICG-001-induced increases in Sftpc and Aqp5, while C/EBPα overexpression alone increased both despite persistent hyperoxia.Conclusions: Canonical Wnt/β-catenin activity and C/EBPα abundance were inversely associated across perturbation experiments in hyperoxia-exposed AT2-like cells, and C/EBPα appears to contribute substantially to the effect of Wnt inhibition on differentiation marker expression. These findings support a functional β-catenin-C/EBPα relationship that may contribute to hyperoxia-associated changes in alveolar differentiation marker expression, pending mechanistic and in vivo confirmation.