Cystic fibrosis (CF) is the most frequent autosomal recessive disease which affects the physiology of the lung. It is due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR protein functions as a Cl- channel.
The most common mutation is a missing phenylalanine at position 508 (delF508) and the associated pathology is a failure of the mutated protein to traffic correctly to the plasma membranes. We showed that anxA5 overexpression increases delF508-CFTR membrane localization. Because anxA5 is increased by GnRH and because PNEC are neuroendocrine cells, our aim is to study their involvement in CF.
Immunochemistry will be used to visualize the GnRH receptor in human lung from normal and CF patients. Tissues will be dissociated and PNEC will be specifically labelled, separated (laser micro-dissection) and cultured. GnRH expression will be assessed in PNEC (RT-PCR, western blotting, immunochemistry). Using surface plasmon resonance the release of GnRH in the culture medium will be assessed. The effect of PNEC upon surrounding airway epithelial cells will be studied: 16HBE14o- (wild type CFTR expressing cells) and CFBE41o- (delF508CFTR expressing cells) will be co-cultured with isolated PNEC either in contact or separated by a permeable membrane. Co-cultured 16HBE14o- and CFBE41o- will be submitted to 2D-Gel electrophoresis. The differentially expressed protein will be identified by mass spectrometry. Finally, the effects of co-cultured PNEC upon CFTR localization and function will be assessed in the epithlial cells.
To apply: Send CV to: pascal.trouve@univ-brest.fr
Expires: July 16, 2010
