SUBSTRATUM-DEPENDENT STIMULATION OF FIBROBLAST MIGRATION BY THE GELATIN-BINDING DOMAIN OF FIBRONECTIN

Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibrob last migration in the transmembrane (or 'Boyden chamber') assay; in co ntrast, the gelatin-binding domain (GBD) of fibronectin has consistent ly been reported to be devoid of migration-stimulating activity in thi s assay, We have examined the effects of fibronectin and several of it s purified functional domains on the migration of human skin fibroblas ts in what is presumably a more physiologically relevant assay involvi ng the movement of cells into a 3-D matrix of native type I collagen f ibrils, We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin , as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femto molar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 mu g/ml), The correct assignment of migration-stimulating activity to GBD, rathe r than to a contaminant, was confirmed by: (a) the use of several fibr onectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed again st epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of na tive fibronectin; and (d) obtaining an identical dose-response curve w ith a genetically engineered GBD peptide, The cryptic migration-stimul ating activity of GBD was not affected by the presence of serum or nat ive fibronectin, but was inhibited by TGF-beta 1, Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migrat ion-stimulating activity in this assay when membranes coated with gela tin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen, Cells preincubated with GBD for 24 hours whilst growing on plastic tissue cu lture dishes and then plated onto native collagen matrices in the abse nce of further GBD also displayed an elevated migration compared to co ntrols, Taken together, these observations suggest that: (a) the inter action of GBD with a putative cell surface receptor (and not the colla gen substratum) initiates a persistent alteration in cell phenotype wh ich is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a h itherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.