Expression of integrin α(v)β3 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-I (MTI-MMP) with tissue inhibitor of MMP-2 (TIMP-2) on the cell surface. Binding of MMP-2 to integrin α(v)β3 has been implicated in presenting activated MMP-2 on the cell surface of invasive cells, but interactions with the MTI-MMP-TIMP-2 system have not been considered. Therefore, we studied the expression and interaction of MTI-MMP, MMP-2 and TIMP-2 in the α(v)β3-negative melanoma cell line BLM and in its β3-transfected, α(v)β3-expressing counterpart BLM-β3, both on cell lines and in xenografts. Total expression levels of MMP-2, MTI-MMP and TIMP-2 did not differ markedly between the α(v)β3- negative and α(v)β3-positive cells. Remarkable differences, however, exist in the presence of active MMP-2 and MTI-MMP. Zymography on cell lysates revealed that active MMP-2 was restricted to α(v)β3-positive cell line and clearly accumulated in xenografts derived from the BLM-β3 cells, confirming the relevance of this integrin for MMP-2 function. Western blotting of cell lysates showed that processing of proMTI-MMP to the activated form was enhanced in BLM-β3. The ratio of active and inactive MTI-MMP was 3-fold higher in the β3-transfectants. Immunofluorescence double-labeling followed by confocal laser microscopy showed co-localization of MTI-MMP and α(v)β3 on BLM-β3 cells, in xenografts from BLM-β3 cells, active MTI-MMP was markedly increased. Our results demonstrate that expression of α(v)β3 in cell lines and xenografts was accompanied by an accumulation of active MTI- MMP and MMP-2. Furthermore, MTI-MMP and α(v)β3 are co-localized on the cell membrane of tumor cells. These findings suggest that activated MTI-MMP co-localized with α(v)β3 may be involved in activation of α(v)β3-bound MMP-2. (C) 2000 Wiley-Liss, Inc.