Advertisement

Pediatric Surgery International

, Volume 29, Issue 1, pp 19–24 | Cite as

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

  • Florian Friedmacher
  • Jan-Hendrik Gosemann
  • Hiromizu Takahashi
  • Nicolae Corcionivoschi
  • Prem Puri
Original Article

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.

Keywords

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

References

  1. 1.
    Gaxiola A, Varon J, Valladolid G (2009) Congenital diaphragmatic hernia: an overview of the etiology and current management. Acta Paediatr 98(4):621–627PubMedCrossRefGoogle Scholar
  2. 2.
    Rollins MD (2012) Recent advances in the management of congenital diaphragmatic hernia. Curr Opin Pediatr 24(3):379–385PubMedCrossRefGoogle Scholar
  3. 3.
    Loane M, Dolk H, Kelly A et al (2011) Paper 4: EUROCAT statistical monitoring: identification and investigation of ten year trends of congenital anomalies in Europe. Birth Defects Res A Clin Mol Teratol 91(Suppl 1):S31–S43PubMedCrossRefGoogle Scholar
  4. 4.
    Keijzer R, Puri P (2010) Congenital diaphragmatic hernia. Semin Pediatr Surg 19(3):180–185PubMedCrossRefGoogle Scholar
  5. 5.
    Doi T, Lukosiute A, Ruttenstock E et al (2010) Disturbance of parathyroid hormone-related protein signaling in the nitrofen-induced hypoplastic lung. Pediatr Surg Int 26(1):45–50PubMedCrossRefGoogle Scholar
  6. 6.
    Ruttenstock E, Doi T, Dingemann J et al (2010) Downregulation of insulin-like growth factor binding protein 3 and 5 in nitrofen-induced pulmonary hypoplasia. Pediatr Surg Int 26(1):59–63PubMedCrossRefGoogle Scholar
  7. 7.
    Mortell A, Montedonico S, Puri P (2006) Animal models in pediatric surgery. Pediatr Surg Int 22(2):111–128PubMedCrossRefGoogle Scholar
  8. 8.
    van Loenhout RB, Tibboel D, Post M et al (2009) Congenital diaphragmatic hernia: comparison of animal models and relevance to the human situation. Neonatology 96(3):137–149PubMedCrossRefGoogle Scholar
  9. 9.
    Beurskens N, Klaassens M, Rottier R et al (2007) Linking animal models to human congenital diaphragmatic hernia. Birth Defects Res A Clin Mol Teratol 79(8):565–572PubMedCrossRefGoogle Scholar
  10. 10.
    Montedonico S, Nakazawa N, Puri P (2008) Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 24(7):755–761PubMedCrossRefGoogle Scholar
  11. 11.
    Sun Q, Jackson RA, Ng C et al (2010) Additional serine/threonine phosphorylation reduces binding affinity but preserves interface topography of substrate proteins to the c-Cbl TKB domain. PLoS One 5(9):e12819PubMedCrossRefGoogle Scholar
  12. 12.
    Thien CB, Langdon WY (2001) Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol 2(4):294–307PubMedCrossRefGoogle Scholar
  13. 13.
    Friedmacher F, Doi T, Gosemann JH et al (2012) Upregulation of fibroblast growth factor receptor 2 and 3 in the late stages of fetal lung development in the nitrofen rat model. Pediatr Surg Int 28(2):195–199PubMedCrossRefGoogle Scholar
  14. 14.
    Teramoto H, Yoneda A, Puri P (2003) Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats. J Pediatr Surg 38(7):1021–1024PubMedCrossRefGoogle Scholar
  15. 15.
    Swaminathan G, Tsygankov AY (2006) The Cbl family proteins: ring leaders in regulation of cell signaling. J Cell Physiol 209(1):21–43PubMedCrossRefGoogle Scholar
  16. 16.
    Takeshita K, Tezuka T, Isozaki Y et al. (2012) Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c. J Biochem [Epub ahead of print]Google Scholar
  17. 17.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682PubMedCrossRefGoogle Scholar
  18. 18.
    Gosche JR, Islam S, Boulanger SC (2005) Congenital diaphragmatic hernia: searching for answers. Am J Surg 190(2):324–332PubMedCrossRefGoogle Scholar
  19. 19.
    Stege G, Fenton A, Jaffray B (2003) Nihilism in the 1990s: the true mortality of congenital diaphragmatic hernia. Pediatrics 112(3 Pt 1):532–535PubMedCrossRefGoogle Scholar
  20. 20.
    Brandsma AE, ten Have-Opbroek AA, Vulto IM et al (1994) Alveolar epithelial composition and architecture of the late fetal pulmonary acinus: an immunocytochemical and morphometric study in a rat model of pulmonary hypoplasia and congenital diaphragmatic hernia. Exp Lung Res 20(6):491–515PubMedCrossRefGoogle Scholar
  21. 21.
    Roth-Kleiner M, Post M (2003) Genetic control of lung development. Biol Neonate 84(1):83–88PubMedCrossRefGoogle Scholar
  22. 22.
    Han RN, Liu J, Tanswell AK et al (1992) Expression of basic fibroblast growth factor and receptor: immunolocalization studies in developing rat fetal lung. Pediatr Res 31(5):435–440PubMedCrossRefGoogle Scholar
  23. 23.
    Sanjay A, Horne WC, Baron R (2001) The Cbl family: ubiquitin ligases regulating signaling by tyrosine kinases. Sci STKE 2001(110):pe40PubMedCrossRefGoogle Scholar
  24. 24.
    Moura RS, Coutinho-Borges JP, Pacheco AP et al (2011) FGF signaling pathway in the developing chick lung: expression and inhibition studies. PLoS One 6(3):e17660PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Florian Friedmacher
    • 1
  • Jan-Hendrik Gosemann
    • 1
  • Hiromizu Takahashi
    • 1
  • Nicolae Corcionivoschi
    • 1
  • Prem Puri
    • 1
    • 2
  1. 1.National Children’s Research CentreOur Lady’s Children’s HospitalDublin 12Ireland
  2. 2.University College Dublin, School of Medicine and Medical ScienceConway Institute of Biomolecular and Biomedical ResearchDublinIreland

Personalised recommendations