BME MS Defense: Nancie Mooney

Adhesion to Cell-Binding Fragments of Fibronectin Determines the Concentration Threshold for Cellular Responses to the Matricryptic Fragment, FNIII1H

Co-Supervised by Professor Denise Hocking

Abstract

The extracellular matrix (ECM) form of fibronectin (FN) stimulates a number of cell behaviors including proliferation, migration, and contractility. As cells deposit FN fibrils into the ECM, FN is placed under cell-derived tension that can expose matricryptic sites, to provide cell signals unique to FN ECM. Fibronectin’s first type III repeat (FNIII1) contains a matricryptic heparin-binding domain. Previous studies have shown that FNIII1C, a fragment of FNIII1 with matricryptic heparin-binding domain exposed, induces G1 phase cell cycle arrest. However, other studies indicate that FNIII1H, a nearly identical fragment of FNIII1, as part of a mimetic protein directly coupled to the cell-binding domain of fibronectin (FNIII8-10), causes an increase in cell proliferation. The mechanism of this dual behavior has not been determined. Using fragments of FNIII8-10 to provide adhesive cell substrates, we examined the effect of the cell-binding domain and matricryptic heparin binding domain on cell behaviors. Addition of FNIII1H in concentrations above a substrate-dependent threshold caused decreases in cell proliferation, cell-substrate contact area, and the number of vinculin-containing focal contacts. The decrease in cell number was due to G1 phase cell cycle arrest, not due to an increase in apoptosis. The substrate-dependent concentration threshold for FNIII1H-induced cell responses was mediated by the length of the cell binding domain fragments as well as the substrate coating concentration. Despite FNIII1H inducing G1 phase cell cycle arrest, the addition of FNIII1H caused an increase in cyclin D1 expression. Addition of FNIII1H reduced Rho activity, while cell adhesion to fragments of fibronectin’s cell binding domain mediated the base levels of Rho activity. In this study, we determined cell-substrate interaction via the cell-binding domain of fibronectin can modify the effects of the matricryptic site of FNIII1 on cell proliferation. These data indicate that the fibronectin’s cell-binding domain-induced cell signals are critical to mediating the cellular effects of the matricryptic site in FNIII1 in full-length fibronectin.