Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/44827 |
| Date | 07 July 2011 |
| Creators | Brown, Ashley Carson |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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