Krüppel-like factor 4 (Klf4) is a novel regulator of neonatal lung fibroblast homeostasis and reduced in hyperoxia-induced lung injury

Abstract

Background: Bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, is characterized by arrest of alveolarization and matrix remodeling. Myofibroblasts are crucial in both processes. Since the transcription factor Kruppel-like factor 4 (Klf4) regulates cell homeostasis and fibroblast function, we studied the functional role of Klf4 in lungs of newborn mice exposed to hyperoxia and in primary neonatal lung fibroblasts (PnF). Methods: (1) Newborn mice were exposed to 85% O2 (HYX) or 21% O2 (NOX) for up to 28 days. (2) Knockdown (siRNA) or overexpression (sleeping beauty transposon system) of Klf4 was induced in PnF or mouse embryonic fibroblasts (MEFs), respectively, followed by exposure to HYX or NOX for up to 48 hours. Results: (1) HYX markedly reduced Klf4 mRNA and protein expression at P7 and P28 in lungs. Gene expression of TGFβ, PAI-1 and αSMA protein (indicator of myofibroblasts) were higher in lungs after HYX. Immunostaining showed a localization of Klf4 in myofibroblasts (αSMA-positive cells). (2) Exposure of PnF to HYX decreased Klf4 protein. Both Knockdown of Klf4 and HYX reduced migration and increased mRNA of CTGF, collagen Iα1 & IVα1 and PDGFRα. Proliferation was not affected by loss of Klf4. In contrast, overexpression of Klf4 in MEFs confirmed the modulation of migration and ECM expression by Klf4 and abrogated the changes induced by HYX. Conclusion: We identify Klf4 as a novel key regulator of neonatal lung fibroblast homeostasis. Loss of Klf4 is intimately linked to myofibroblast activation, fibrosis and migration, eventually reducing thereby alveolar formation and contributing to the pathogeneis of BPD.