CELL-MIGRATION PROMOTED BY A POTENT GRGDS-CONTAINING THROMBIN-CLEAVAGE FRAGMENT OF OSTEOPONTIN

Osteopontin (OPN) is a secreted adhesive glycoprotein with a gly-arg-g ly-asp-ser (GRGDS) cell binding domain. Several independent studies ha ve suggested that OPN functions in tumor growth and metastasis, and on e likely possibility is that OPN facilitates tumor invasion by promoti ng tumor cell migration. Consistent with this hypothesis, immobilized OPN promoted concentration-dependent tumor cell migration (i.e., hapto taxis) in modified Boyden chambers. In particular, cleavage of OPN by thrombin, which likely occurs in the tumor microenvironment, resulted in enhancement of OPNs haptotactic activity; and assays performed with purified preparations of the two individual OPN thrombin-cleavage fra gments demonstrated that all detectable activity was associated with t he GRGDS-containing fragment. In contrast to the activity of both OPN and its GRGDS-containing fragment in promoting haptotaxis, neither of these proteins in solution promoted chemotaxis, indicating that each m ust be immobilized to promote cell migration. In haptotaxis assays, an tibody LM609 to integrin alpha(v) beta(3) blocked > 80% cell migration towards the GRGDS-containing OPN fragment, implicating alpha(v) beta( 3) as its principal functional receptor. In comparison with equimolar quantities of other adhesive proteins, the GRGDS-containing OPN thromb in-cleavage fragment was not only > 2-fold more effective than intact OPN at promoting haptotaxis, but also > 8-fold and > 6-fold more effec tive than fibrinogen and vitronectin, respectively, indicating that th is OPN fragment is highly active relative to other alpha(v) beta(3) li gands.