The activation of ProMMP-2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MT1-MMP (MMP-14) to a 45 kDa form.

We have assessed the effect of fibronectin and laminin-1 on the expression of molecules involved in the activation pathway of MMP-2, a key proteinase in tissue remodelling. HT1080 fibrosarcoma cells cultured on fibronectin were shown to activate endogenous MMP-2, to a level comparable with that elicited by treatment with phorbol ester. In contrast, the MMP-2 expressed by HT1080 cells cultured on laminin-1 was mainly in the pro- (inactive form). Culture of the cells on peptide fragments of fibronectin derived from the central cell binding domain also promoted MMP-2 activation, indicating that signals via fibronectin binding to integrin receptors may be involved. HT1080 cells cultured on immobilised antibodies to the alpha5 and beta1 integrin subunits secreted levels of active MMP-2 similar to those observed for full length fibronectin, whereas cells cultured on an antibody to the alpha6 integrin subunit secreted mainly proMMP-2. The data demonstrate that the activation of MMP-2 by HT1080 cells is regulated by the nature of the extracellular matrix, and that signals via the alpha5beta1 integrin receptor may be involved in the fibronectin induced up-regulation of MMP-2 activation. We then assessed the effect of fibronectin on the components of the putative MT1-MMP/TIMP-2 'receptor' complex implicated in MMP-2 activation. Levels of TIMP-2 protein expressed by HT1080 cells did not vary detectably between cells cultured on fibronectin or laminin-1. However, the expression of MT1-MMP protein was up-regulated when the cells were cultured on fibronectin, which could be attributed to an increase in levels of a truncated 45 kDa form. Parallel studies using gelatin zymography demonstrated that the up-regulation of the production of the 45 kDa band was concomitant with MMP-2 activation. Inhibitor studies revealed that the truncation of MT1-MMP to a 45 kDa form is MMP mediated, although not inhibited by TIMP-1. In vitro, the 45 kDa form could be generated by cleavage of membrane-bound native MT1-MMP with several recombinant MMPs, including both active MT1-MMP and MMP-2. The implication that either MMP-2 or MT1-MMP can process MT1-MMP to 45 kDa, raises the possibility that truncation of MT1-MMP represents a self-regulatory end-point in the activation pathway of MMP-2.