Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells

Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endoth elioma cells. The current study was designed to (a) characterize the presen ce of MMPs in the vascular tumor, (b) define whether these MMPs originate f rom the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endoth elial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24.24) an d MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis o f the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell line s, suggesting that stroma cells, particularly endothelial cells recruited b y the tumor cells, rather than eEnd.1 cells themselves, are the source of t he gelatinases observed in the tumors in vivo. The conditioned medium of eE nd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagen ase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. Th e increased production of MMP-2 and MMP-1, observed at the protein level (z ymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was n ot prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that re gulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothe lial cells, the stimulation of MMPs can also occur at the level of secretio n, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis. (C) 2000 Academic Press.