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CREB1 signalling in the developing mouse lung
thesisposted on 2017-01-09, 01:38 authored by Antony, Nisha
Transcription factors play a crucial role in regulating cell type specific gene expression programs which influence branching morphogenesis, cellular proliferation and differentiation during fetal lung development. The Cyclic AMP Response Element-Binding Protein 1 (Creb1) transcription factor mediates cyclic adenosine 3',5'-monophosphate (cAMP) signalling in a range of cell types. The aims of these studies were to investigate the specific role of cAMP signalling via Creb1 in the developing mouse lung using both in vivo and in vitro experiments. For in vivo studies three transgenic mice models were used, these were mice with complete loss of Creb1 (Creb1-/-), mice with lung epithelial cell-specific and mesenchymal cell-specific loss of Creb1. Chapter 3 of this thesis characterised two lipogenic gene targets, stearoyl-CoA desaturase 1 (Scd1) and fatty acid synthase (Fasn) which were found in reduced levels in mice lung with complete and epithelial cell-specific loss of Creb1. We hypothesized that Creb1 plays a crucial role in the transcriptional regulation of lipid biosynthetic pathways in lung epithelial cells during late gestation. In this study Scd1 and Fasn were shown to be down regulated in the E17.5 Creb1-/- mouse lung; while there was an increase in the mRNA levels of lipogenic transcription factors SrebpF1, C/ebpα and Ppar-Gamma. Studies using gene-targeted mice with germ line and conditional deletion of Creb1 in lung epithelial cells showed an absence of Scd1 in type II AECs, with little effect on Fas expression. In vitro studies in the mouse MLE-15 lung epithelial cell line showed Creb1 to be essential for maintaining Scd1 gene expression. Lipid profiling showed that blocking Creb1 activity in MLE-15 cells by transfection of a dominant-negative Creb1 vector suppressed desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal lung epithelial cells. Chapters 4 and 5 examined the cell type specific role of Creb1 in the developing lung mesenchymal versus epithelial cells using two Cre recombinase mediated Creb1-deleted mouse lines; a doxycycline inducible SPCrtTA/(TetO)7Cre mouse line to delete Creb1 in the respiratory epithelial cells and a Dermo1Cre mouse line to delete Creb1 in mesenchymal cells. Fetal mice with epithelial-specific deletion of Creb1 were born at the expected mendelian frequency. Histological analysis revealed abnormal dilation of saccules in the distal airways during the saccular stage (E17.5). Postnatally the lung at P14 appeared to have regions with thicker alveolar walls. Expression of surfactant proteins and epithelial markers were all reduced at both the mRNA and protein level. Surfactant protein C staining was detectable in the distal regions; however there was a localized loss in the abnormally dilated saccules. Mice with mesenchyme specific Creb1 deficiency were viable and born at the expected mendelian frequency. The lung appeared normal at the early and late saccular stage of development. In conclusion the studies in this thesis indicate an essential role of Creb1 in lung epithelial cell differentiation at the saccular stage in mice during late gestation.