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A6114 - Krüppel-Like Factor 4 (Klf4) Regulates ATII Cell Survival and Is Decreased in Lungs of Newborn Mice Exposed to Hyperoxia
Author Block: J. Mohr1, M. Koch2, N. Oikonomou3, K. Dinger1, S. Wickström4, C. Vohlen1, D. Hirani1, M. Odenthal5, J. Dötsch6, M. Alejandre Alcazar1; 1Department of Pediatrics, Translational Experimental Pediatrics - Experimental Pulmonology, University Hospital Cologne, Cologne, Germany, 2Center for Biochemistry, University of Cologne, Cologne, Germany, 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, 4Skin Homeostasis and Ageing, Paul Gerson Unna Research Group, Max Planck Institute for Biology of Ageing, Cologne, Germany, 5Institute of Pathology, University Hospital Cologne, Cologne, Germany, 6Department of Pediatrics, University Hospital Cologne, Cologne, Germany.
RATIONALE Preterm neonates exposed to mechanical ventilation (MV) and/or increased O2 evolve bronchopulmonary dysplasia (BPD), a neonatal chronic lung disease, characterized by reduced alveolar formation. Prior studies showed that MV reduces Krüppel-like factor 4 (Klf4), a transcription factor that regulates cell pluripotency and survival. Since type II alveolar epithelial cells (ATII cells) are crucial in alveolarization and serve as progenitor cells for lung regeneration, we hypothesized: Klf4 is expressed in ATII cells during late lung development; dysregulation of Klf4 is mechanistically linked to reduced alveolarization after exposure to increased O2 or MV.
METHODS 1) Neonatal C57BL/6N mice were exposed to 85% O2 or room air from postnatal day 1 (P1) until P28. Lungs were excised and either snap-frozen or pressure-fixed with paraformaldehyde, followed by paraffin embedding. 2) Murine ATII cells (MLE-12) were exposed to either hyperoxia (85% O2) vs normoxia (21% O2) or to cyclic stretch (20% elongation; 1Hz). Klf4 was overexpressed in MLE-12 using a doxycycline-inducible sleeping beauty transposon system. Survival was assessed by MTT or BrdU assay.
RESULTS 1) Dual immunofluorescent staining for Klf4 and surfactant protein C (SfptC) showed localization of Klf4 in ATII cells during late lung development (P7 and P28). Exposure of newborn mice to hyperoxia decreased gene expression and protein abundance of Klf4 in lungs significantly. The loss of Klf4 was linked to reduced mRNA and protein of alveolar cell-specific markers (SftpC and Aquaporin 5), indicating arrested alveolar growth. Reduced radial alveolar count coupled with increased mean linear intercept confirmed less alveolar formation after hyperoxia. 2) Hyperoxia did not only decrease Klf4 protein and mRNA expression in cultured MLE-12, but also inhibited cell survival (MTT and BrdU). Moreover, prolonged exposure of MLE-12 cells to cyclic stretch for 24 hours reduced Klf4 mRNA. Intriguingly, overexpression of Klf4 did not protect MLE-12 cells from reduced survival after hyperoxia; in contrast, Klf4 overexpression aggravated the adverse effect of hyperoxia on proliferation of MLE-12 cells, indicating thereby that both loss and overexpression of Klf4 regulate ATII cell homeostasis.
CONCLUSIONS We show for the first time, that Klf4 is expressed in ATII cells during late lung development. Loss of Klf4 in vitro an in vivo following hyperoxia was linked to reduced survival and lung growth, respectively. Here, we identify Klf4 as a novel key regulator of ATII cell homeostatasis and a potential novel target to prevent hyperoxia-induced lung injury.