Hyperoxia-induced neonatal rat lung injury involves activation of TGF-{beta} and Wnt signaling and is protected by rosiglitazone

Abstract

Despite tremendous technological and therapeutic advances, bronchopulmonary dysplasia (BPD) remains a leading cause of respiratory morbidity in very low birth weight infants, and there are no effective preventive and/or therapeutic options. We have previously reported that hyperoxia-induced neonatal rat lung injury might be prevented by rosiglitazone (RGZ). Here, we characterize 1) perturbations in wingless/Int (Wnt) and transforming growth factor (TGF)-beta signaling, and 2) structural aberrations in lung morphology following 7-day continuous in vivo hyperoxia exposure to neonatal rats. We also tested whether treatment of neonatal pups with RGZ, concomitant to hyperoxia, could prevent such aberrations. Our study revealed that hyperoxia caused significant upregulation of Wnt signaling protein markers lymphoid enhancer factor 1 (Lef-1) and beta-catenin and TGF-beta pathway transducers phosphorylated Smad3 and Smad7 proteins in whole rat lung extracts. These changes were also accompanied by upregulation of myogenic marker proteins alpha-smooth muscle actin (alpha-SMA) and calponin but significant downregulation of the lipogenic marker peroxisome proliferator-activated receptor-gamma (PPARgamma) expression. These molecular perturbations were associated with reduction in alveolar septal thickness, radial alveolar count, and larger alveoli in the hyperoxia-exposed lung. These hyperoxia-induced molecular and morphological changes were prevented by systemic administration of RGZ, with lung sections appearing near normal. This is the first evidence that in vivo hyperoxia induces activation of both Wnt and TGF-beta signal transduction pathways in lung and of its near complete prevention by RGZ. Hyperoxia-induced arrest in alveolar development, a hallmark of BPD, along with these molecular changes strongly implicates these proteins in hyperoxia-induced lung injury. Administration of PPARgamma agonists may thus be a potential strategy to attenuate hyperoxia-induced lung injury and subsequent BPD.

Fig. 1.

A: rosiglitazone (RGZ) prevented hyperoxia-induced aberrations in lung architecture. Seven-day continuous hyperoxia (95% O₂) exposure alone caused failure of secondary septation of distal lung air sacs resulting in larger than normal alveoli denoted by asterisks, with marked decrease in interstitial thickness (arrows). These changes were prevented by administration of RGZ (1 and 3 mg/kg ip) concomitant to hyperoxia exposure. Representative hematoxylin-eosin-stained lung sections are shown (magnification, ×40; bar, 20 μm; n = 6). B: morphometric changes of lungs in hyperoxia without and with RGZ treatment. RGZ prevented 7-day hyperoxia (95% O₂)-induced decrease in lung alveolar septal thickness, radial alveolar count, alveolar tissue density, and the accompanying increase in mean linear intercept. Significant changes in all of these parameters were observed on exposure to 95% hyperoxia alone, which were prevented by RGZ treatment (*P < 0.05, 95 vs. 21% O₂ and **P < 0.05, 95% O₂ + RGZ vs. 95% O₂ only; n = 6). C: visualization of neutrophils influx in lung of rat pups exposed to 95% O₂. Representative myeloperoxidase antibody-stained lung sections (5 μm) are presented. Myeloperoxidase-reactive neutrophils (with characteristic polylobulated nucleus) are seen in hyperoxic alveoli, which are absent in normoxic and RGZ treated lungs. Magnification, ×1,000; arrows point to the myeloperoxidase reactive neutrophils; calibration bar = 20 μm; n = 4.

Fig. 2.

A: concomitant administration of 3 mg/kg RGZ inhibited hyperoxia (95% O₂ for 7 days)-induced changes in α-smooth muscle actin (α-SMA) and calponin expression. α-SMA and calponin were upregulated in hyperoxia, and RGZ administration inhibited the hyperoxia-induced upregulation of these proteins. Representative protein bands in duplicate and densitometric histogram of each group are shown (*P < 0.05, 95 vs. 21% O₂, and **P < 0.05, 95% O₂ + 3 mg/kg RGZ vs. 95% O₂ only; n = 8). B: concomitant administration of 3 mg/kg RGZ inhibited hyperoxia (95% O₂ for 7 days)-induced changes in peroxisome proliferator-activated receptor-γ (PPARγ), lymphoid enhancer factor 1 (Lef-1), and β-catenin in whole lung protein extract of 7-day-old rat pups. Seven-day hyperoxia exposure resulted in a significant decrease in PPARγ and a significant increase in Lef-1 and β-catenin protein expression, and concomitant RGZ administration prevented all of these changes. Representative protein bands in duplicate and densitometric histogram of each group are shown (*P < 0.05, 95 vs. 21% O₂, and **P < 0.05, 95% O₂ + 3 mg/kg RGZ vs. 95% O₂ only; n = 8). AU, arbitrary units.

Fig. 3.

A and B: immunostaining for lung Lef-1 and lipoprotein receptor-related protein 6 (LRP6) in neonatal rat pups exposed to 21% O₂, 95% O₂, or 95% O₂ + 3 mg/kg RGZ for 7 days. Representative Lef-1 and LRP6 antibody-stained lung sections (5 μm) are shown. Top: magnification, ×400, with insets. Bottom pictures are inset regions of top; magnification, ×1,000 for Lef-1 and ×600 for LRP6. Arrows point to the lung regions showing Lef-1 and LRP6 staining. At 21% O₂ exposure, lung sections have weak base line Lef-1 and LRP6 expression, which is robustly upregulated with 95% O₂ exposure. RGZ (3 mg/kg) administration during 95% O₂ exposure prevented this upregulation for both Lef-1 and LRP6 (n = 4).

Fig. 4.

A and B: administration of RGZ inhibited hyperoxia-induced changes in phosphorylated Smad3 (pSmad3) and Smad7 protein levels in whole lung protein extract of 7-day-old rat pups. Although there were no significant perturbations in the total Smad3 levels on exposure to hyperoxia, pSmad3 and Smad7 levels increased significantly. Activin receptor-like kinase 5 (ALK-5) protein level also increased significantly on with hyperoxia (B). Concomitant RGZ treatment prevented hyperoxia-induced increases in pSmad3 and Smad7 protein levels but not that of ALK-5. Representative protein bands in duplicate and densitometric histogram of each group are shown (*P < 0.05, 95 vs. 21% O₂, and **P < 0.05, 95% O₂ + 3 mg/kg RGZ vs. 95% O₂ only; n = 8).

Fig. 5.

A and B: confirmation of hyperoxia-induced activation of pulmonary transforming growth factor (TGF)-β and wingless/Int (Wnt) signaling in TOPGAL mice. Seven-day exposure to 95% O₂ to TOPGAL mice starting on postnatal day 1 resulted in clear evidence of Wnt and TGF-β signaling activation. A shows bluish staining (LacZ positivity) of lung tissue at both macroscopic and microscopic levels. Hyperoxia exposure resulted in increased expression of ALK-5, pSmad3/total Smad3 (T-Smad3), Lef-1, α-SMA, and fibronectin protein levels, clearly indicating activations of TGF-β and Wnt signaling pathways (B; P < 0.05 vs. 21% O₂; n = 4).

Fig. 6.

Evidence for hyperoxia-induced activation of TGF-β and Wnt signaling in alveolar interstitial fibroblasts. Exposure of cultured rat lung alveolar interstitial fibroblasts to 24-h hyperoxia (95% O₂) with or without RGZ (10 μM) or anti-TGF-β (10 μg/ml medium) pretreatment resulted in significant increases in pSmad3 (30 min) and Lef-1 (24 h) protein levels, accompanied by a significant decrease in phosphorylated (p-) β-catenin (30 min) protein level. Pretreatment with either anti-TGF-β antibody or RGZ blocked hyperoxia-induced increases in pSmad3 and Lef-1 and a decrease in p-β-catenin (*P < 0.05, 95 vs. 21% O₂, and **P < 0.05, 95% O₂ + RGZ or 95% O₂ + anti-TGF-β vs. 95% O₂ only; n = 4). For pSmad3 and p-β-catenin analysis, cells were concomitantly also treated with calyculin A (50 nM).

Fig. 7.

Evidence for interactions between the TGF-β, Wnt, and PPARγ signaling pathways in alveolar interstitial fibroblasts in response to hyperoxia. Immunoprecipitation of whole cell lysates of alveolar interstitial fibroblasts following 24-h exposure to either normoxia or hyperoxia (95% O₂), with or without RGZ (10 μM) with anti-β-catenin antibody, and subjecting these complexes to Western blot analysis using anti-Smad3, anti-Lef-1, and anti-PPARγ antibodies showed that hyperoxia resulted in significant decreases in β-catenin-bound Smad3 and Lef-1, whereas levels of β-catenin-bound PPARγ increased. Treatment with RGZ both before and during hyperoxia exposure resulted in attenuation against hyperoxia-induced decreases in Smad3 and Lef-1 bound to β-catenin, whereas the level of β-catenin-bound PPARγ increased further (*P < 0.05, 95 vs. 21% O₂, and **P < 0.05, 95% O₂ + RGZ vs. 95% O₂ only; n = 4).