Regulation of urokinase plasminogen activator/plasmin- mediated invasion of melanoma cells by the integrin vitronectin receptor αVβ3

Abstract

The integrin vitronectin receptor αvβ3 is a mediator of cellular migration and invasion and has been identified as a marker of progression in malignant melanoma. Using a human melanoma model, we have previously shown that this receptor was coordinately expressed with the receptor for the urokinase plasminogen activator (uPAR). In our present study, the link between these receptors was further investigated by assessing the effect of αvβ3 ligation on uPAR transcription and function. Using the reverse transcription-polymerase chain reaction, we found that receptor ligation by immobilized monoclonal antibodies (MAbs) induced a rapid increase (up to 4.5 fold) in uPAR mRNA levels, which was maximal 4 hr after cell attachment. An increase was also noted in plasminogen activator inhibitor type-1 (PAI-1) mRNA levels (2.7-fold), but none was noted in uPA levels. In addition, ligation of αvβ3 resulted in a significant increase in cell surface-associated plasmin levels, which coincided with a 2- to 3-fold increase in cell invasion as measured in the Matrigel invasion assay. This increase in invasion could in turn be abolished by antibodies directed to uPA and uPAR and by the plasmin inhibitors ϵ-aminocaproic acid and aprotinin. Furthermore, ligation of the integrin αvβ3 triggered a rapid increase of up to 12-fold in total cellular PKC activity, and this coincided with the redistribution of PKCβ, but not PKCα, from the cytosol to the membrane. Treatment of the cells with the PKCβ-specific inhibitor LY379196 blocked uPAR and PAI-1 mRNA induction and reduced the increase in cell invasion due to αvβ3 ligation, confirming the involvement of this isoform in the response. The results provide evidence that the vitronectin receptor can enhance invasion by regulating the uPAR/uPA/plasmin system of proteolysis and implicate PKCβ as an intermediate in the activation pathway. © 2001 Wiley-Liss, Inc.