The role of the Foxo transcription factors in normal and aberrant late lung development

Abstract

Bronchopulmonary dysplasia is a common complication of the lung in premature babies characterized by decreased septation, alveolar hypoplasia, less surface area for gas exchange and dysmorphic vascular development. The Foxo transcription factors are a family of proteins accreditated with a pivotal role in the regulation of the development of the mouse and the pathogenesis of lung diseases such as cancer and pulmonary hypertension. In a hyperoxia mouse model of BPD (85% O2), the steady-state mRNA levels of Foxo1 were significantly downregulated. The Foxo4 mRNA levels were partially reduced, Foxo3 and Foxo6 levels were upregulated. Newborn mice exposed to hyperoxia from the day of birth to the fourteenth day of life revealed a significant downregulation of the phosphorylated form of Foxo1 at S256. Hyperoxia induced protein and mRNA reduction of Foxo1 at P14. The protein expression of Foxo4 was significantly downregulated in hyperoxia. The assessment of the gene expression of the Foxo transcription factors in a cell culture-based experiment has shown a significantly higher expression of Foxo1 in lung endothelial cells compared with AT2 cells, PFBs and PASMCs. In endothelial cells FACS-sorted from the lung of P14 mice exposed to hyperoxia, Foxo1 expression was higher in endothelial cells compared to the other cell types considered. In P14 mice exposed to hyperoxia, Foxo1 mRNA levels were downregulated only in endothelial cells. Flow cytometry analysis of microvascular endothelial cells from newborn mice exposed to hyperoxia revealed a significant decrease in the apoptosis levels assessed by AnnexinV and a significant decrease in the number of endothelial cells compared with the age-matched controls. The conditional downregulation of Foxo1 gene expression in endothelial cells of newborn mice documented that the reduction of Foxo1 is crucially involved in the significant reduction of the number of endothelial cells and body mass of P14 mice. Bulk RNA-seq analysis of microvascular endothelial cells FACS-sorted from the lung of newborn mice exposed to hyperoxia showed the downregulation of genes centrally involved in the apoptotic signalling such as Parp1, Gadd45b and E2f1. The conditional downregulation of Foxo1 in lung endothelial cells of newborn mice induced the downregulation of Parp1, Ccne1 and E2f1. The assessment of the lung structure by stereological analysis showed that the conditional downregulation of Foxo1 in endothelial cells induced arrested lung development in a way comparable with the simplification of the lung structure induced by hyperoxia. The conditional upregulation of Foxo1 in the endothelial cells of newborn mice exposed to hyperoxia rescued alveolarization from hyperoxia-induced lung injury. Nab-Paclitaxel administration of 10 μg/g on P4 and P10 activated the total amount of Foxo1 protein and the phosphorylation of Foxo1 at S256 but not the gene expression of Foxo1 in newborn wildtype mice. Stereological analysis revealed that Nab-Paclitaxel partially preserved the lung structure from hyperoxic damage. To date, this is the first study providing detailed insight into the role of Foxo1 as an important inductor of alveolarization during late lung development and a crucial modulator of the apoptotic signallings in lung endothelial cells in the contest of hyperoxia-induced lung damage. These data provide a clear picture of the function of Foxo1 not only as an important mediator of alveolarization during late lung development but also as an indispensable inducer of apoptosis in lung endothelial cells during mouse development.