Association of the proliferation of lung fibroblasts with the ERK1/2 signaling pathway in neonatal rats with hyperoxia-induced lung fibrosis

Abstract

Bronchopulmonary dysplasia (BPD) is a common, serious complication occurring in premature infants. Although clinical characteristics and pathologic changes are well described, the pathogenesis of alveolar dysplasia and interstitial fibrosis is less clear. Lung fibroblasts (LFs) are present in the extracellular matrix and serve essential roles during pulmonary epithelial injury and in response to fibrosis development in BPD. The current study investigated hyperoxia-induced proliferation of primary LFs in vitro and mechanisms that may be involved. Newborn rats were exposed to 90% oxygen, while control rats were kept in normal atmosphere. Primary LFs were isolated on postnatal day 3, 7 and 14. Hyperoxia-induced proliferation of LFs isolated on day 7 and 14 by accelerating the cell cycle progression from G1 to S phase. Collagen type I protein secretion and mRNA expression on day 7 and 14 were increased by hyperoxia compared with the controls. Hyperoxia significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and significantly increased collagen type I expression compared with the room air control group. The findings indicated that an increase in LF proliferation in response to hyperoxia was associated with ERK1/2 phosphorylation. This mechanism may contribute to over-proliferation of LFs leading to disturbed formation of normal alveoli.

Keywords: extracellular signal-regulated kinase; fibrosis; hyperoxia; lung fibroblast.

Figure 1.

Lung morphology. (A) Hematoxylin and eosin staining of lung tissues at postnatal day 3, 7 and 14. In the room air group, alveolar formation was gradually completed with lung development following birth. In the hyperoxia group, the following manifestations were observed over time: Decreased number of alveoli, dilation of terminal alveolar spaces, simplification of alveoli and pulmonary fibrosis. Scale bar, 100 µm. (B) RAC values were significantly lower in the hyperoxia group compared with the room air group starting from postnatal day 7 onwards (n=5). (C) Lung tissue fibrosis scores over time. Higher scores were determined in the hyperoxia group compared with the room air group starting at postnatal day 7 (n=5). *P<0.05, **P<0.01. RAC, radial alveolar count.

Figure 2.

Identification of LFs and proliferation assays. (A) Immunocytochemical staining of LFs from control rats. Primary LFs were stained with vimentin. LFs exhibited stellate morphology, with an elongated spindle-like structure. Scale bar, 100 µm. (B) Immunofluorescence staining of LFs. Primary LFs from control rats were labeled with vimentin-specific antibodies (green) and cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 50 µm. (C) Cell-cycle distribution. Cell populations in G0/G1, S and G2/M phases were determined by calculating the mean of five independent experiments. The proportion of cells in the G0/G1 phase decreased, associated with an increase in the S phase in LFs from neonatal rats in the hyperoxia group at postnatal day 7 and 14 compared with the room air control group (n=5). (D) Effect of hyperoxia on cell proliferation. CCK-8 assays were used to measure proliferation. Hyperoxia promoted cell proliferation at postnatal day 7 and 14 (n=6). (E) Col-I secreted protein levels in LFs determined by ELISA. An increase was observed in the hyperoxia group compared with the room air control group (n=5). *P<0.05, **P<0.01. LF, lung fibroblast; Col-I, collagen type I; CCK-8, cell counting kit-8; OD, optical density.

Figure 3.

Phosphorylation of ERK1/2 in lung tissue samples. (A) Increased p-ERK1/2 antigen levels were observed in the cytoplasm and nucleus of pulmonary epithelial and mesenchymal cells by immunohistochemistry using the peroxidase-conjugated streptavidin method. Scale bar, 50 µm. (B) Protein levels of p-ERK were detected by immunohistochemistry. p-ERK levels increased in the hyperoxia group starting from postnatal day 7 compared with the room air control group (n=5). (C) Western blot analysis of lung tissue samples detecting p-ERK1/2, ERK1/2 and β-actin. (D) Western blot quantification suggested that the intergroup difference was more significant at postnatal day 7 and 14 for p-ERK levels (n=5). **P<0.01. ERK, extracellular signal-regulated kinase; p, phosphorylation.

Figure 4.

Phosphorylation of ERK1/2 in LFs. (A) Immunocytochemistry was performed in LFs to detect were the expression levels of p-ERK1/2. Increased p-ERK1/2 levels were detected in the cell cytoplasm and nucleus in the hyperoxia groups at postnatal day 7 and 14. Scale bar, 50 µm. (B) p-ERK levels were detected by immunohistochemistry. p-ERK levels increased in the hyperoxia group starting from postnatal day 7 compared with the room air control group (n=5). (C) Western blot analysis of the LFs detecting p-ERK1/2, ERK1/2 and β-actin. (D) Quantification of western blots. Following hyperoxic exposure levels of phosphorylated ERK1/2 increased on day 7 and 14 compared with the room air control (n=5). **P<0.01. ERK, extracellular signal-regulated kinase; p, phosphorylation; LF, lung fibroblast.