Expression of integrin alpha(v)beta(3) correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo

Abstract

Activation of matrix metalloproteinase-2 (MMP-2) is mediated by binding to the complex of membrane-type matrix metalloproteinase-1 (MT1-MMP) with tissue inhibitor of MMP-2 (TIMP-2) on the cell surface. Binding of MMP-2 to integrin alpha(v)beta(3) has been implicated in presenting activated MMP-2 on the cell surface of invasive cells, but interactions with the MT1-MMP-TIMP-2 system have not been considered. Therefore, we studied the expression and interaction of MT1-MMP, MMP-2 and TIMP-2 in the alpha(v)beta(3)-negative melanoma cell line BLM and in its beta(3)-transfected, alpha(v)beta(3)-expressing counterpart BLM-beta(3), both on cell lines and in xenografts. Total expression levels of MMP-2, MT1-MMP and TIMP-2 did not differ markedly between the alpha(v)beta(3)-negative and alpha(v)beta(3)-positive cells. Remarkable differences, however, exist in the presence of active MMP-2 and MT1-MMP. Zymography on cell lysates revealed that active MMP-2 was restricted to alpha(v)beta(3)-positive cell line and clearly accumulated in xenografts derived from the BLM-beta(3) cells, confirming the relevance of this integrin for MMP-2 function. Western blotting of cell lysates showed that processing of proMT1-MMP to the activated form was enhanced in BLM-beta(3). The ratio of active and inactive MT1-MMP was 3-fold higher in the beta(3)-transfectants. Immunofluorescence double-labeling followed by confocal laser microscopy showed co-localization of MT1-MMP and alpha(v)beta(3) on BLM-beta(3) cells. In xenografts from BLM-beta(3) cells, active MT1-MMP was markedly increased. Our results demonstrate that expression of alpha(v)beta(3) in cell lines and xenografts was accompanied by an accumulation of active MT1-MMP and MMP-2. Furthermore, MT1-MMP and alpha(v)beta(3) are co-localized on the cell membrane of tumor cells. These findings suggest that activated MT1-MMP co-localized with alpha(v)beta(3) may be involved in activation of alpha(v)beta(3)-bound MMP-2.