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Spatiotemporal Changes In Sprouty-2 Signaling and Tyrosine Phosphorylation In Nitrofen-Induced Pulmonary Hypoplasia

Sunday, October 21, 2012: 7:54 AM
Versailles Ballroom (Hilton Riverside)
Florian Friedmacher1, Jan-H. Gosemann1, Naho Fujiwara1, Nicolae Corcionivoschi1, Luis A. Alvarez2 and Prem Puri1, (1)Our Lady's Children's Hospital, National Children's Research Centre, Dublin, Ireland, (2)University College Dublin, Conway Institute of Biomedical Research, Dublin, Ireland

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.