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Regulation of integrin adhesions by varying the density of substrate-bound epidermal growth factor.

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.

SUBMITTER: Shahal T

PROVIDER: S-EPMC3939687 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundChemotropic factors in the extracellular microenvironment guide nerve growth by acting on the growth cone located at the tip of extending axons. Growth cone extension requires the coordination of cytoskeleton-dependent membrane protrusion and dynamic adhesion to the extracellular matrix, yet how chemotropic factors regulate these events remains an outstanding question. We demonstrated previously that the inhibitory factor myelin-associated glycoprotein (MAG) triggers endocytic removal of the adhesion receptor ?1-integrin from the growth cone surface membrane to negatively remodel substrate adhesions during chemorepulsion. Here, we tested how a neurotrophin might affect integrin adhesions.ResultsWe report that brain-derived neurotropic factor (BDNF) positively regulates the formation of substrate adhesions in axonal growth cones during stimulated outgrowth and prevents removal of ?1-integrin adhesions by MAG. Treatment of Xenopus spinal neurons with BDNF rapidly triggered ?1-integrin clustering and induced the dynamic formation of nascent vinculin-containing adhesion complexes in the growth cone periphery. Both the formation of nascent ?1-integrin adhesions and the stimulation of axon extension by BDNF required cytoplasmic calcium ion signaling and integrin activation at the cell surface. Exposure to MAG decreased the number of ?1-integrin adhesions in the growth cone during inhibition of axon extension. In contrast, the BDNF-induced adhesions were resistant to negative remodeling by MAG, correlating with the ability of BDNF pretreatment to counteract MAG-inhibition of axon extension. Pre-exposure to MAG prevented the BDNF-induced formation of ?1-integrin adhesions and blocked the stimulation of axon extension by BDNF.ConclusionsAltogether, these findings demonstrate the neurotrophin-dependent formation of integrin-based adhesions in the growth cone and reveal how a positive regulator of substrate adhesions can block the negative remodeling and growth inhibitory effects of MAG. Such bidirectional remodeling may allow the growth cone to rapidly adjust adhesiveness to the extracellular matrix as a general mechanism for governing axon extension. Techniques for manipulating integrin internalization and activation state may be important for overcoming local inhibitory factors after traumatic injury or neurodegenerative disease to enhance regenerative nerve growth.

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Regulation of integrin adhesions by varying the density of substrate-bound epidermal growth factor.

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