Decreased pulmonary c-Cbl expression and tyrosine phosphorylation in the nitrofen-induced rat model of congenital diaphragmatic hernia

  • Friedmacher F
  • Gosemann J
  • Takahashi H
 et al. 
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Abstract

{PURPOSE}

The high morbidity of newborn infants with congenital diaphragmatic hernia ({CDH)} is attributed to pulmonary hypoplasia ({PH)}, which is characterized by a failure of alveolar development. 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 is essential for a proper lung development, is disrupted during late gestation of nitrofen-induced {CDH.} Casitas B-lineage lymphoma (c-Cbl) proteins are known regulators of signal transduction through {FGFRs}, indicating their important role during alveolarization in developing lungs. Furthermore, it has been demonstrated that tyrosine phosphorylation of c-Cbl proteins has a pivotal role for their physiological function and activity during fetal lung development. We designed this study to test the hypothesis that pulmonary c-Cbl expression and tyrosine phosphorylation status are decreased in the nitrofen-induced {CDH} model.


{METHODS}

Timed-pregnant rats received either 100 mg nitrofen or vehicle on gestation day 9 (D9). Fetuses were harvested on D18 and D21, and lungs were divided into two groups: control and hypoplastic lungs with {CDH} ({CDH(+))} (n = 10 at each time-point, respectively). Pulmonary gene expression levels of c-Cbl were analyzed by quantitative real-time polymerase chain reaction. Western blotting combined with densitometry analysis was used for semi-quantification of protein levels of pulmonary c-Cbl and tyrosine phosphorylation status. Confocal-immunofluorescence staining was performed to evaluate c-Cbl protein expression and distribution.


{RESULTS}

Relative {mRNA} expression levels of pulmonary c-Cbl were significantly decreased in {CDH(+)} on D18 and D21 compared to controls. Western blotting showed markedly decreased protein levels of pulmonary c-Cbl and tyrosine phosphorylation status in {CDH(+)} on D18 and D21. Confocal-immunofluorescence analysis confirmed decreased c-Cbl expression in {CDH(+)} on D18 and D21 mainly in the distal alveolar epithelium compared to controls.


{CONCLUSION}

Decreased pulmonary c-Cbl gene and protein expression accompanied by a decreased tyrosine phosphorylation status during the late stages of fetal lung development may result in reduced c-Cbl activity, and thus interfere with the {FGFR-mediated} alveolarization in the nitrofen-induced {CDH} model.

Author-supplied keywords

  • Congenital diaphragmatic hernia
  • Fetal lung development
  • Nitrofen
  • Tyrosine phosphorylation
  • c-Cbl

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