Cooperation between plasmin and membrane-type 1 metalloproteinase (mt1-mmp) in the activation of 72 kda type iv collagenase (mmp-2) on the cell surface

Extracellular matrix degradation requires a proteinase cascade that involves components of the plasminogen activator-plasmin system and matrix metalloproteinases (MMPs). All MMPs are secreted as inactive zymogens and are activated extracellularly by limited proteolysis, a fundamental step in the control of their activity. We have recently reported that plasmin activates cellbound proMMP-2 (72 kDa type IV collagenase, or gelatinase A) but degrades it in soluble phase. The cell surface appears to play an important role in the regulation of this proteinase cascade. Urokinase binding to its receptor in the presence of plasminogen potently accelerates pro-MMP-2 activation. Binding of plasmin to the cell surface is also required, as E-aminocaproic acid inhibits proMMP-2 activation. The membrane-type MMP (MT1-MMP), a transmembrane MMP that binds MMP-2HTMP-2 (tissue inhibitor of metalloproteinases-2) complex on the cell membrane, has been implicated as a physiological activator of proMMP-2. However, high levels of MT1-MMP are required for proMMP-2 activation, suggesting that other factors) may be involved in the physiological processing of proMMP-2. To characterize potential interactions between MTI-MMP and components of the urokinaseplasmin system, we studied proMMP-2 activation in human HT1080 fibrosarcoma cells transfected with MTI-MMP antisense cDNA. Cells transfected with the empty vector, which expressed significant amounts of MTIMMP, or with the antisense cDNA, which expressed no MTI-MMP, secreted MMP-2 only in its proenzyme form. Whereas all cells expressing MTI-MMP had cell-associated MMP-2, cells transfected with antisense MTI-MMP had no membrane-bound MMP-2. Addition of plasminogen (4 ug/ml) to vectortransfected cells resulted in the conversion of proMMP-2 into its 62 kDa activation product. This effect was inhibited by aprotinin but not by metalloproteinase inhibitors, indicating that the catalytic activity of MTI-MMP is not required for plasmin activation of MMP-2. Addition of plasminogen to cells transfected with the antisense cDNA, which expressed no MTI-MMP and had no cell-associated MMP-2, failed to activate proMMP-2, showing that proMMP-2 binding to MTI-MMP is required for gelatinase activation. Thus, plasmin appears to be a physiological activator of MTl-MMP-bound proMMP-2.