Cell Surface Proteolysis by Serine Proteinases Enhances RGD-Sensitive Melanoma Cell Adhesion on Fibrinogen and Vitronectin

Abstract

Tumor cells avidly secrete various proteinases, and cascades of proteolytic activation occur around the cells. Therefore, cell surface receptors of tumor cells are under the constant influence of proteinases. In this study, the effects of serine proteinases on integrin-mediated cell-matrix interactions were studied in C32TG and Mewo human melanoma cells. These melanoma cells were pretreated with proteinases and their adhesive properties on various substrata were evaluated by cell adhesion assays. Paradoxically, appropriate cell surface proteolysis enhanced the RGD-sensitive cell adhesion on fibrinogen and vitronectin, but not the RGD-insensitive adhesion on type I collagen or laminin. Pretreatment of these cells with 0.1 to 1 μM of trypsin, chymotrypsin, or plasmin for 30 min at 37°C increased the number of spread cells on fibrinogen and vitronectin by 200-300%. The enhancement of cell spreading was not accompanied by up-regulation of the relevant RGD-sensitive integrin expression. Analysis of the cell surface receptor by GRGDSPK-Sepharose affinity chromatography showed that trypsin treatment did not up-regulate αvβ3 integrin, an RGD-sensitive receptor for fibrinogen and vitronectin in the melanoma cells, nor the induced appearance of novel receptors. Treatment of cells with 100 nM proteinases increased cell binding of both monoclonal and polyclonal antibodies against αvβ3 integrin subunits by 70%, but not that of monoclonal antibody against α2, α3, or α6 subunit, indicating that cell surface proteolysis exposed more αvβ3 integrin on the cell surface. These results suggest that exposure of αvβ3 integrin is a part of the mechanisms underlying the serine proteinase-induced enhancement of melanoma cell adhesion on fibrinogen and vitronectin.