AbstractAirway branching and vascular growth in the developing lung are intimately co-ordinated processes which involve cross-talk between the primary inducer of airway tube elongation, Fibroblast Growth Factor 10 (FGF10) and the pro-vasculogenic transcription factor, Hypoxia Inducible Factor 1a (HIF-1a). The FGF receptor antagonist, Sprouty2 (SPRY2), influences the duration of this signalling activity and thus governs periodical airway and vascular branching. However, the mechanism behind this coordination is unknown. The aim of this thesis was to establish if SPRY2 acts as a key co-ordinator of airway and vascular development in the fetal lung and to explore the implications of this role for vasculogenic signalling from the endodermal epithelium.
SPRY2 is present in the nucleus in both rat Fetal Distal Lung Epithelial (FDLE) and Human Bronchial Epithelial (HBE) cells where its FGF10-induced loss correlates with an increase in Histone 3 (H3) phospho-acetylation. Chromatin Immunoprecipitation (ChIP) assays revealed a dominant interaction between SPRY2 and GC-rich regions of the Vascular Endothelial Growth Factor (VEGF-A) promoter. Mutation of SPRY2 at tyrosine 55 (Y55) and SH3 motifs induced HIF-1a transcriptional activity while by contrast; cysteine mutation suppressed this activity and abolished SPRY2 interaction with the VEGF-A promoter. Knockdown of SPRY2 by shRNA induced phosphorylation of Extracellular Signal-regulated Kinases 1 & 2 (ERK1/2) and Mammalian Target of Rapamycin Complex 1 (mTORC1) while also amplifying VEGF-A gene expression. Knockdown of SPRY2 amplified HIF-1a transcriptional activity approximately 3.5 fold at normoxia and this was reflected in ChIP assays showing increased association of HIF-1a with the VEGF-A promoter.
Taken as a whole, this data supports the hypothesis that SPRY2 is potentially the ‘missing link’ responsible for co-ordinated airway and vascular growth of the lung epithelium. It achieves this by regulating vascular gene expression by binding to methylation sites of the VEGF-A promoter, coupled with the potential for SPRY2 to bind G-proteins to its SH3 binding domain in order to silence airway and potentially vascular signalling cues from the epithelium.
|Date of Award||2013|
|Supervisor||Stephen Land (Supervisor) & Marios Stavridis (Supervisor)|