AN INVESTIGATION INTO THE ROLE OF MMP9 IN REGULATING CYTOSKELETAL REMODELLING DURING TGF-β INDUCED EMT IN THE LENS
| Autor: | Liu, Zi Zhen (Ginny) |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Druh dokumentu: | Diplomová práce |
| Popis: | Fibrotic cataracts are attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT) (Eldred et al., 2011). Using in vivo and ex vivo mouse knockout (KO) models, our laboratory has identified matrix metalloproteinase-9 (MMP9) as an essential protein in the EMT process. However, alternations to the cytoskeleton were observed in MMP9KO mouse lens epithelial cells (LECs) (Korol et al., 2014). In addition, alpha-smooth muscle actin and filamentous actin stress fibres were absent in TGF-β2 treated MMP9KO mouse LECs, and a NanoString analysis revealed no marked differences in ACTA2 and ACTB expression between the lenses of TGF-β-overexpressing mice (TGF-βtg) and TGF-βtg mice on a MMP9KO background. Our laboratory subsequently conducted a cytoskeletal protein array, which revealed the differential regulation of numerous proteins in MMP9KO mouse LECs. The purpose of this thesis is to validate select proteins from the protein array, and reveal pathways that are regulated by MMP9 during TGF-β2-induced EMT. In order to improve the efficiency of experiments, the novel MMP9-specific inhibitor, JNJ0966, was confirmed to be able to prevent TGF-β2-induced EMT in rat LEC explants. Cortactin, focal adhesion kinase (FAK), lim-domain kinase 1 (LIMK1) and myosin light chain 2 were selected from the array and analyzed via western blot and immunofluorescence analyses. Results from the protein array and validation studies agree for all selected proteins except for FAK. Although FAK was upregulated in rat LECs that were co-treated with JNJ0966 and TGF-β2 (TG:JNJ), it was not activated to exert its effects. Interestingly, LIMK1 was also observed to be localized to the nucleus in JNJ0966-treated LECs. The localization of MRTF-A was also analyzed via immunohistochemistry, and we observed reduced MRTF-A nuclear translocation in TG:JNJ LECs. Results from this thesis revealed that MMP9 deficiency differentially regulated proteins involved in actin polymerization and cell migration, and these alterations conferred resistance against TGF-β2-induced EMT. Thesis Master of Science (MSc) |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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