The objective of this proposal is to develop new data showing that the homeobox gene, Hoxb-5, is necessary for normal cellular and matrix interactions during lung development, and that aberrant cell interactions controlled by Hoxb-5 expression are involved in the development of congenital lung malformations, BPS and CCAM. Previously, it has been shown that the """"""""master regulatory gene"""""""" Hoxb-5 is necessary for normal airway branching and that miss-expression of Hoxb-5 alters proximal airway branching during early lung morphogenesis. The applicant has studied the developmental expression pattern of Hoxb-5 in human lung, and it was found that its expression suggests a role in the regulation of bronchial airway branching morphogenesis, similar to the findings in the mouse. The applicants have recently shown that Hoxb-5 is abnormally present in BPS and CCAM tissue mimicing an earlier period of lung development. The hypotheses of the proposed work is that Hoxb-5 controls normal airway branching morphogenesis through regulation of cell adhesion and extracellular matrix (ECM) molecules, and that abnormal airway development in BPS and CCAM involves mis-regulated Hoxb-5 expression.
Specific Aim #1 will test the hypothesis that normal Hoxb-5 expression helps establish airway branching patterns and epithelial cell fate by regulating the site-specific expression of cell adhesion and ECM molecules. Using gestational day 11.5 embryonic mouse lung cultures, Hoxb-5 expression will be stimulated with retinoic acid or inhibited with antisense oligonucleotides. Airway branching patterns and terminal bud counts will be measured. The effects on quantitative and site-specific changes in the expression of molecules involved in cell adhesion (E-Cadherin, integrin) and cell-matrix interactions (laminin, tenascin) will be studied using quantitative competitive polymerase chain reaction (QC-PCR), in situ hybridization, inimunoprecipitation and immunocytochemistry. The effects of altered Hoxb-5, E-Cadherin, integrin, laminin and tenascin expression on proximal versus distal epithelial cell fate will be studied by in situ hybridization and immunocytochemistry of Clara Cell lOkD (CC1O) protein and surfactant protein C (SPC).
Specific Aim #2 will show that BPS and CCAM lesions express abnormally elevated levels of Hoxb-5 in association with aberrant changes in cell adhesion and ECM molecules involved in lung branching morphogenesis. Using Western blot analysis and immunocytochemistry, quantitative and site-specific expression of Hoxb-5, cell adhesion (E-Cadherin and integrin) and ECM (laminin and tenascin) molecules will be studied in BPS and CCAM tissue and compared to expression in normal human lung tissue to determine if abnormal Hoxb-5 expression in BPS and CCAM is associated with changes in cell adhesion and ECM molecules. These studies will help decipher the mechanisms involved in normal lung morphogenesis and show how these mechanisms are altered in BPS and CCAM. These studies will also help develop a mechanistic in vitro model to test how these processes are regulated. An understanding of the mechanisms involved in the development of congenital lung anomalies will permit the development of novel therapies to reduce prenatal and postnatal morbidity and mortality related to these abnormalities.
