| Summary: | Thesis (M.Sc.)--Memorial University of Newfoundland, 2009. Medicine Includes bibliographical references (leaves 79-95) Research Question: Transforming growth factor beta (TGF-β) causes growth stimulation and transformation in fibroblasts, but growth inhibition/apoptosis in other cell types. Previously, TGF-β has been shown to activate the Smad signalling cascade in all cell types. Alternative signalling pathways have been described in response to TGF-β. To explain how TGF-β controls growth, we investigated a downstream target of phosphatidylinositol 3-kinase (PI3K) called Akt. Akt inactivates glycogen synthase kinase 3 beta (GSK-3β) and FOXO. We propose that PI3K signalling is partially responsible for the different phenotypic effects of TGF-β in mesenchymal and epithelial cells. -- Methods: Western blotting was used to describe temporal changes (0-3 hours) in PDK-1, Akt, GSK-3β and Cyclin Dl phosphorylation/protein levels with TGF-β2 stimulation of normal fibroblast and epithelial cell lines. Additionally, we blocked the effects of TGF-β on Akt/GSK-3β using PI3K/Akt specific inhibitors and TGF-β receptor kinase dead cell lines. Using the UAS/GAL4 system of ectoptic gene expression in Drosophila melanogaster, we overexpressed the activated Baboon receptor (Activin/TGF-β type I homologue) in an eye-specific manner, using early and late developmental drivers. We also coexpressed GSK-3β, PI3KDN and Akt constructs with activated Baboon. Results were documented using Scanning Electron Microscopy (SEM). -- Results: TGF-β stimulation increased PDK-1, Akt and GSK-3β phosphorylation in fibroblasts over time. Conversely, TGF-β stimulation decreased PDK-1, Akt and GSK-3β phosphorylation over time in epithelial cells. The GSK-3β substrate, Cyclin Dl, exhibited decreasing phosphorylation in fibroblast cells, and decreased protein levels of Cyclin Dl in epithelial cells. In fibroblasts, TGF-β increased Cyclin Dl complex formation with CDK-4 and nuclear localization. Conversely, TGF-β decreased complex formation in epithelial cells. TGF-β stimulated Cyclin Dl/CDK complex formation was blocked by a PI3K inhibitor. Morphological transformation by TGF-beta was attenuated by an Akt inhibitor in fibroblasts. In Drosophila studies, overexpression of constitutively active Baboon in the eye caused overgrowth and patterning defects that was rescued by coexpressing either a PI3KDN mutation, mutant dFOXO or mutant GSK-3β, while overexpression of wild-type Akt or PI3K had a synergistic effect. -- Summary: These results, in both cell culture and Drosophila, point to a novel mechanism of TGF-β signaling through PI3K/Akt. In total, these results define a novel pathway for TGF-β regulation of cell cycle through PI3K/Akt in normal cells. This signalling pathway also differentiates epithelial and fibroblast cell responses to TGF-β stimulation. In conjunction with Smads, this pathway could account for the cell type specific growth responses to TGF-β. Potential alterations of this pathway may be a mechanism by which some cancers elude the normal growth regulatory system imposed by TGF-β.
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