Lipoxin A4 Attenuates Bronchopulmonary Dysplasia via Upregulation of Let-7c and Downregulation of TGF-β1 Signaling Pathway

Abstract

Transforming growth factor-β (TGF-β) superfamily members are key regulators for lung development and progress of bronchopulmonary dysplasia (BPD). The mechanisms by which lipoxin A₄ (LXA₄) attenuates development of BPD have not been clarified. Neonatal murine BPD models were inducted by hyperoxia treatment. Neonatal mice were exposed to room air or 85% O₂ hyperoxia with or without treatment with 5S,6R-methyl-LXA₄ or anti-TGF-β antibodies. Mouse lung epithelial cells (MLE-12 cells) and mouse embryonic fibroblasts (NIH/3T3 cells) were cultured in room air or 85% O₂ followed by treatment of LXA₄, anti-TGF-β antibodies, and let-7c mimic/anti-microRNA transfections. Treatment with 5S,6R-methyl-LXA₄ and anti-TGF-β antibodies both attenuated the mice alveolar simplification induced by hyperoxia. Hyperoxia treatment significantly altered pulmonary basal mRNA and protein expressions of several important extracellular matrix (ECM) and ECM remodeling proteins including fibronectin, α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), elastin, tenascin C, collagen I, and matrix metalloproteinase-1 (MMP-1). 5S,6R-methyl-LXA₄ and anti-TGF-β antibodies suppressed the mRNA and protein expressions of TGF-β₁ and TGF-βR1 but not TGF-βR2 in the lungs exposed to hyperoxia. Treatment with LXA₄ and anti-TGF-β antibodies alleviated hyperoxia-induced injury of the NIH/3T3 cells identified by morphologic observation and flow cytometry, and expressions of ECM, ECM remodeling proteins, and TGF-β₁ signaling pathway, but reversed by transfection with let-7c anti-miRNA. LXA₄ upregulated the let-7c expression in MLE-12 cells, transfection with let-7c anti-miRNA, inhibited the LXA₄-induced let-7c expression in MLE-12 cells exposed to hyperoxia and reduced the relative luciferase activity of let-7c binding with let-7c binding sites of the TGF-βR1 3' UTR. Treatment with 5S,6R-methyl-LXA₄ and anti-TGF-β antibodies significantly improved histology, ECM, and ECM remodeling proteins in the lungs isolated from the murine BPD model induced by hyperoxia. The LXA₄-imparted protective effects on hyperoxia-induced lung injury are mediated by upregulation of let-7c and inhibition of TGF-β₁ and subsequent downregulation of TGF-β₁ signaling pathway.

Keywords: bronchopulmonary dysplasia; let-7; lipoxin A4; transforming growth factor-β.