Purpose: Pulmonary hypoplasia (PH) remains the most life-threatening cause in newborn infants with congenital diaphragmatic hernia (CDH). The nitrofen-induced CDH model has been widely used to investigate the pathogenesis of PH in CDH. It has previously been shown that the fibroblast growth factor receptor (FGFR) pathway, which plays a key role in fetal lung development, is disrupted during alveologenesis in nitrofen-induced PH. In fetal lungs, Sprouty-2 (SPRY-2) functions as a novel regulator of FGFR signaling and is mainly expressed by epithelial cells. SPRY-2 knockouts showed severe defects in lung morphogenesis identical to nitrofen-induced PH, indicating the important role in developing fetal lungs. Furthermore, it has been demonstrated that SPRY-2 is tyrosine-phosphorylated upon stimulation by FGFR, suggesting that tyrosine phosphorylation activates SPRY-2, which is essential for its physiological function during lung development. We designed this study to investigate the hypothesis that SPRY-2 signaling and tyrosine phosphorylation are altered in nitrofen-induced PH.
Methods: Time-pregnant rats received either 100 mg nitrofen or olive oil on day 9 of gestation (D9) (Ethics: B100/4378). Fetuses were harvested on D18 and D21, and fetal lungs were divided into 3 groups: control, hypoplastic lungs without CDH (CDH-), and hypoplastic lungs with CDH (CDH+) (n=8 at each time-point, respectively). Pulmonary gene expression levels of SPRY-2 were analyzed by quantitative real-time polymerase chain reaction (RT-PCR). Western blotting was performed to evaluate pulmonary protein level of SPRY-2 and tyrosine phosphorylation patterns. Immunohistochemistry was used to investigate SPRY-2 expression and distribution.
Results: Relative mRNA expression levels of pulmonary SPRY-2 were significantly decreased in CDH- and CDH+ on D18 compared to controls (*P<0.05) (Figure 1A). Western blotting revealed markedly decreased pulmonary SPRY-2 expression between D18 and D21 (Figure 1B). Tyrosine phosphorylation (p-Tyr) was decreased in CDH- and CDH+ on D18 and absent on D21 (Figure 1B). Immunohistochemistry confirmed markedly decreased SPRY-2 expression in CDH- and CDH+ on D18 and D21 mainly in the distal epithelium compared to controls (Figure 1C).
Conclusion: Spatiotemporal changes in pulmonary SPRY-2 gene and protein expression and loss of tyrosine phosphorylation during the late stages of fetal lung development may result in decreased SPRY-2 activity and thus interfere with the FGFR-mediated alveologenesis in the nitrofen-induced CDH model.