Abstract
Substrates coated with specific bioactive ligands are important for tissue engineering, enabling the local presentation of extracellular stimulants at controlled positions and densities. In this study, we examined the cross-talk between integrin and epidermal growth factor (EGF) receptors following their interaction with surface-immobilized Arg-Gly-Asp (RGD) and EGF ligands, respectively. Surfaces of glass coverslips, modified with biotinylated silane-polyethylene glycol, were functionalized by either biotinylated RGD or EGF (or both) via the biotin-NeutrAvidin interaction. Fluorescent labeling of the adhering A431 epidermoid carcinoma cells for zyxin or actin indicated that EGF had a dual effect on focal adhesions (FA) and stress fibers: at low concentrations (0.1; 1 ng/ml), it stimulated their growth; whereas at higher concentrations, on surfaces with low to intermediate RGD densities, it induced their disassembly, leading to cell detachment. The EGF-dependent dissociation of FAs was, however, attenuated on higher RGD density surfaces. Simultaneous stimulation by both immobilized RGD and EGF suggest a strong synergy between integrin and EGFR signaling, in FA induction and cell spreading. A critical threshold level of EGF was required to induce significant variation in cell adhesion; beyond this critical density, the immobilized molecule had a considerably stronger effect on cell adhesion than did soluble EGF. The mechanisms underlying this synergy between the adhesion ligand and EGF are discussed.
Highlights
This study focuses on the cross-talk between an adhesive (RGD) and a signaling (EGF) molecule, and its impact on cell–matrix adhesion
It was shown that integrin clustering is essential for cellsurface attachment, spreading and focal adhesions (FA) formation [12, 13]; attachment and spreading of A431 epidermoid carcinoma cells were investigated on surfaces functionalized with varying RGD densities
It was previously shown that the addition of epidermal growth factor (EGF) to the growth medium of serum-starved cells results in a significant growth of FAs, and the associated actin stress fibers [10]
Summary
This study focuses on the cross-talk between an adhesive (RGD) and a signaling (EGF) molecule, and its impact on cell–matrix adhesion. EGF is a growth factor present in a soluble form. It has been shown that the EGF receptor (EGFR) and its downstream signaling cascade are activated only when integrin receptors are aggregated and activated by their respective adhesion ligands [2]. The cross-talk between these two ligand-receptor systems affects the reciprocal process; namely, integrin-mediated adhesion [3,4,5]. The addition of soluble EGF to the epidermoid carcinoma cells, A431 and other cell types resulted in the formation of aligned actin stress fibers [3, 6,7,8]. Growth and elongation of focal adhesions along
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