Krüppel-like Factor 4 is a Stat3-Smad2 regulator in ATII cells determining ATII cell fate during lung development and hyperoxia-induced lung injury

Background: Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolarization and regeneration. Kruppel-like factor 4 (Klf4), a transcription factor regulating cell homeostasis and pluripotency, is highly expressed in human and mice during lung development and linked to lung growth arrest. Aim: To study (1) if hyperoxia-induced lung injury reduces Klf4 in ATII cells in mice; (2) if ATII-specific loss of Klf4 depletes number of ATII cells; (3) the function of Klf4 in ATII cells in vitro. Methods: (1) Newborn mice were exposed to 85% O2 (HYX) or room air (NOX). (2) Cell-specific deletion of Klf4 was induced in mice during late lung development. (3) Overexpression (Klf4OE) and ablation of Klf4 (Klf4del; CRISPR/Cas9) in murine lung epithelial cells (MLE-12). Results: (1) Dual immunofluorescent staining determined Klf4 in ATII cells during late murine lung development and in epithelial cells in the human fetal lung. Reduced ATII markers after HYX were linked to reduced Klf4 expression and lung growth. (2) ATII-specific Klf4 ablation during late lung development reduced ATII cells in mice. (3) In MLE-12, HYX decreased Klf4 expression and cell survival. Klf4OE and Klf4del in MLE-12 induced apoptosis and decreased epithelial cell markers. Klf4OE aggravated HYX-related decreased proliferation, activated Smad2 and blocked Stat3 signaling. Inhibition of Smad2, using an inhibitor, activated Stat3. Klf4del activated Stat3 and inhibited Smad2. Conclusion: We identified a novel Klf4-Smad2-Stat3 axis cell homeostasis that HYX disrupts in ATII cells and in lungs of newborn mice, suggesting Klf4 as potential target regulating lung regeneration.