Interactions between fibronectin fragments, proteases and the β6 integrin in the control of epithelial cell behaviour
Epithelial cells interact with their surrounding extracellular matrix via integrin receptors. Signals generated in this way control epithelial cell migration, proliferation and apoptosis. Changes in the extracellular matrix, integrin expression and protease secretion occur in wound healing and cancer and thus these systems are thought to play a crucial role in such processes. We hypothesise that the interactions between these key players affect epithelial cell behaviour. We have utilised a variety of epithelial cells including normal primary cells and lines with manipulated integrin expression to investigate this further. Epithelial cells are commonly grown in medium containing additives such as hydrocortisone, EGF, insulin and cholera toxin. When epithelial cell are cultured in medium without additives they adopt a more mesenchymal phenotype. This was shown to be due to the absence of EGF which resulted in a down-regulation of the cell-cell adhesion molecule e-cadherin. Experiments were performed exarnining the interaction of epithelial cells with a variety of fibronectin fragments. The adhesion of normal keratinocytes and cell lines expressing low (CI) and high (VB6) levels of the p6 integrin subunit was unaffected on the 120kDa fibronectin fragment when compared to the full length molecule. Motility has been followed using wound assays and Transwell migration assays. The latter showed that migration was significantly increased on the 120kDa fragment but only in the VB6 cells. This may be achieved through the up-regulation of matrix metalloproteinase 9 (MMP-9) which is increased in the supernatant from VB6 cells plated on 120kDa fragment In vitro this fragment can be generated by incubating full length fibronectin with purified MMP-9. Immunohistochemical studies using oral squamous cell carcinoma tissue indicate that these interactions may occur in vivo with fibronectin, β6 integrin and MMPs all being located at the invading tumour front. We have thus identified an important feedback loop where expression of the p6 integrin subunit which is only seen in wound healing and cancer enhances the motility of and MMP secretion by epithelial cells in contact with the 120kDa fibronectin fragment These enzymes can men further degrade fibronectin to generate additional fragments. If it were possible to distinguish this fragment from the parent molecule immunologically it could prove to be a valuable marker of aggressive OSCC being indicative of a number of pro-tumourigenic processes.