Cell Signalling and Stress Fiber Formation

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Transformation of polarized epithelial cells to non-polarized mesenchymal cells, also known as epithelial to mesenchymal transition (EMT) is a biological process that imparts cells with enhanced capabilities of migration, evasion of apoptosis, and production of extracellular matrix components. Due to these features, EMT is known to play a critical role in numerous biological mechanisms including embryogenesis and pathologies such as cancer and fibrosis. During EMT transformed cells show extensive cytoskeletal remodeling including the acquisition of stress fibers composed of F-actin and the contractile protein a-smooth muscle actin (aSMA). These EMT events are often induced by the ligands such as epithelial growth factor and transforming growth factor beta (TGFß). The model that we have used to investigate stress fiber formation and EMT is the rodent ocular lens. The ocular lens is a unique and powerful model in that it is an avascular organ consisting of only two cell types, lens epithelial cells and lens fiber cells, permitting easy isolation and culturing of a pure epithelial cell population. Additionally, when lens epithelial cells undergo EMT and stress fiber formation they loose their normal transparent nature, which is a physiological response that can be measured quantitatively. Using lens epithelial cell (LEC), whole lens and in vivo rodent models our lab made the observation that in the absence of matrix metalloproteinase 9 (MMP9), an enzyme that degrades the extracellular matrix, LEC loose their ability to form TGFß-induced stress fibers and undergo EMT. From these findings, and additional preliminary results, we hypothesize that MMP9, plays a critical role in cytoskeletal remodeling and stress fiber formation during EMT. We will further explore this question using our established models involving the excised rat lens and rat/mouse lens explants. We will also employ our in vivo approaches including adenoviral delivery of TGFß (AdTGFß) and transgenic/knockout mouse models, all of which are unique to our laboratory. Our specific aims are: 1. To determine the cell signaling molecules dependent on MMP9 in TGFß-induced stress fiber formation and EMT. 2. To investigate how MMP9 and required targets revealed in Aim 1 impact stress fiber formation and EMT. 3. To determine if restoration of MMP9 expression, or expression of candidate targets (from Aim 1) will cause LEC to form stress fibers and undergo EMT. Together these experiments will provide a collective effort to reveal the MMP9-mediated signaling pathways necessary for LEC EMT and associated changes in the cell cytoskeleton. Findings generated will support our long-term goal to understand the signaling cascade(s) required for regulating cytoskeletal remodeling during EMT as it pertains to numerous biological events, both normal and pathological.