Hyperoxia modulates TGF-β/BMP signaling in a mouse model of bronchopulmonary dysplasia

Abstract

-Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-␤ and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O2 between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O2, and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-␤ type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-␤ signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O2 as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O2. After exposure to 85% O2, primary alveolar type II cells were more susceptible to TGF-␤-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O2 significantly enhanced the TGF-␤-stimulated production of the ␣1 subunit of type I collagen (I␣1), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-␤/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Glucocorticoids Recruit Tgfbr3 and Smad1 to Shift Transforming Growth Factor-β Signaling from the Tgfbr1/Smad2/3 Axis to the Acvrl1/Smad1 Axis in Lung Fibroblasts

“…Primary human lung fibroblasts were obtained from Lonza. Primary mouse lung fibroblasts were isolated as described previously (22). The NIH/3T3 mouse fibroblast-like cell line (CRL-1658 TM ) and H441 human Clara cell-like airway epithelial cell line (HTB-174 TM ) were obtained from the American Type Culture Collection.…”

“…Protein concentration was determined by Bradford assay. Proteins (25 g/lane) were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and immunoblots were probed as described previously (22), using the following antibodies: mouse anti-rabbit Tgfbr3 (Cell Signaling Technology; 2519; 1:1000), mouse anti-rabbit ␤-actin (Cell Signaling Technology; 4967; 1:1000), mouse anti-rabbit phospho-Smad1/5/8 (Cell Signaling Technology; 9511; 1:800), mouse anti-rabbit Smad1 (Cell Signaling Technology; 9743; 1:1000), mouse anti-rabbit phospho-Smad2 (Cell Signaling Technology; 3101; 1:1000), mouse anti-rabbit phospho-Smad3 (Cell Signaling Technology; 9520; 1:1000), mouse anti-mouse Smad2 (Cell Signaling Technology; 3103; 1:1000), mouse anti-rabbit Smad2/3 (Cell Signaling Technology; 3102; 1:1000), rabbit anti-bovine MYH11 (smooth muscle myosin heavy chain 11; Abcam, ab53219; 1:1000), and monoclonal mouse anti-rabbit ACTA2 (␣-smooth muscle actin; Sigma, A-2547; 1:1000). Immune complexes were detected using peroxidase-conjugated secondary antibodies: anti-rabbit (ThermoFisher Scientific; rb:13460; 1:3000) and anti-mouse (ThermoFisher Scientific; ms:31450; 1:3000).…”

“…Real Time RT-PCR Analysis-Total RNA was harvested from cell cultures or mouse lung tissue, after homogenization as described for immunoblotting, using the PeqGold total RNA kit (Peqlab; 12-6834-01) and screened by quantitative real time RT-PCR with the primers listed in Table 1, as described previously (22,44,45). Changes in mRNA expression were assessed using the gapdh gene as a reference, as described previously (22,44,45).…”

“…In lung fibrosis, the pro-fibrotic activities of TGF-␤ drive the production of the fibrotic mediator connective tissue growth factor and plasminogen activator inhibitor-1 (encoded by the SERPINE1 gene in humans) by lung fibroblasts and promote fibroblast to pathological myofibroblast differentiation (16). TGF-␤ also drives aberrant production of extracellular matrix molecules such pro-collagen, which limit alveolar repair and proper alveolar development in diseases such as chronic obstructive pulmonary disease (17)(18)(19), lung fibrosis (17,20), acute respiratory distress syndrome (21), and bronchopulmonary dysplasia (22,23). In general, matrix production relies on TGF-␤ signaling by the type I TGF-␤ receptor Tgfbr1 (also called , in complex with the type II receptor (Tgfbr2), which together recruit the downstream signaling molecules Smad2 and Smad3 to transduce signals to the nucleus, regulating the expression of TGF-␤-responsive genes in the so-called "Tgfbr1/Smad2/3 axis" (24 -26).…”

Effects of Preterm Birth on the Kidney

“…Although the levels of supplemental oxygen administered to the preterm infant have been substantially reduced in recent years, the levels of oxygen in the bloodstream remain elevated above normal; hence it is important that research is conducted into the effects of hyperoxia on nephrogenesis in the preterm infant. Certainly, experimental studies in the lung have shown that hyperoxia can lead to the generation of oxygen free radicals, infiltration of inflammatory cells, collagen deposition, cellular apoptosis and subsequent tissue injury (McGrath-Morrow and Stahl, 2001;Dieperink et al, 2006;Alejandre-Alcazar et al, 2007;Chen et al, 2007;Chetty et al, 2008). Whether this is also the case in other tissues has not been examined.…”