Abstract
BackgroundSecondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation.MethodsMice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/Go to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27kip1 and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27kip1 in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry.ResultsIn neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27kip1 correlated with reduced LF entry into S-phase.ConclusionsNuclear localization of FoxO3a, an important regulator of p27kip1 gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.
Highlights
Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness
We observed that the proportion of Platelet-derived growth factor receptor (PDGFR)-expressing lung fibroblasts (LF) which contain nuclear p27kip1 is significantly higher at P12, whereas this proportion does not change in the remaining alveolar cells, which lack the green fluorescent protein (GFP)-marker of PDGFRα-gene expression and whose nuclei only exhibit staining with PoPo3 (Figure 1)
These data indicate that PDGFRα-mediated signaling may regulate the intracellular location of p27kip1 and that regulation changes during septal formation
Summary
Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. During the period of maximal secondary alveolar septal elongation, which extends from postnatal day 4 (P4) through day 14 in mice, the proliferation of interstitial lung fibroblasts (LF) declines from its initial maximum. PDGF-A, and its exclusive signaling through PDGFRα, are absolutely required for secondary alveolar septal formation [2]. PDGFRα-expressing LF preferentially localize to the distal portion of the septa, contain more abundant ACTA2 (α-smooth muscle actin, αSMA), and are required for elastic fiber formation [3]. The 3H-thymidine autoradiographic labeling index of interstitial LF was maximal at P4 and declined afterwards, and cells nearest the origin of the secondary septal crests were more likely to proliferate [4]. The labeling index of lipid-laden LF declines earlier than for LF which lack lipid droplets [6]. The proliferation of LF which contain αSMA, and more abundantly express PDGFRα, declines earlier than for LF which do not express PDGFRα [7]
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