Connective tissue growth factor enhances keratinocyte adhesion to fibronectin and promotes migration through integrin alpha5/beta1

Abstract

Introduction: We have previously shown that connective tissue growth factor (CTGF) promotes keratinocyte migration during re-epithelialization. In this study, we investigated whether the CTGF-driven migration involved integrin alpha-5/beta-1 - the principal ligand for fibronectin (FN).Methods: Adhesions assays were performed by coating wells with 10 ug/mL FN or phosphate buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/mL CTGF, 5 mmol/L EDTA, 10 mmol/L Mg2+, 10 ug/mL anti-integrin alpha-5/beta-1-blocking antibody. Chemotaxsis assays were performed using a modified Boyden chamber. Keratinocytes were pre-incubated with alpha-5/beta-1-antibodies or mouse-IgG for 30 minute, and migration in the absence or presence of 200 ng/mL CTGF was measured. Cells were stained and absorbance was measured at 570 nm. A value of 1 was assigned to untreated cells.Results: Cell adhesion increased 1.5 ± 0.3 folds in wells coated with FN compared to PBS. CTGF enhanced cell adhesion 2.1 ± 0.3 folds, while EDTA reduced CTGF mediated cell adhesion to baseline (1.1 ± 0.2). The addition of the divalent cation Mg2+ restored CTGF-induced adhesion, indicating involvement of integrins. Integrin alpha-5/beta-1-blocking antibodies reversed CTGF-enhanced binding (1.1 ± 0.2). Consistent with the cell adhesion data, CTGF-induced migration was reduced to 1.5 ± 0.3 by anti-integrin alpha-5/beta-1 antibodies compared to the 2.0 ± 0.6 fold increase seen with 200 ng/mL CTGF.