PROTEOLYTIC PROCESSING OF MEMBRANE-TYPE-1 MATRIX METALLOPROTEINASE ISASSOCIATED WITH GELATINASE-A ACTIVATION AT THE CELL-SURFACE

Human fibroblasts and MT-1080 fibrosarcoma cells express membrane-type -1 matrix metalloproteinase (MT1-MMP), the cell surface activator of g elatinase A, in separate forms of 63 kDa, 60 kDa and in some cases 43 kDa. In the present work the interrelationships between MTI-MMP proces sing and gelatinase A activation were analysed using MT-1080 fibrosarc oma cells as a model. It was found that MTI-MMP was synthesized as a 6 3 kDa protein, which was constitutively processed to a 60 kDa active e nzyme with N-terminal Tyr(112), as shown by immunoprecipitation, immun oblotting and sequence analyses. Co-immunoprecipitation results indica ted that only the active 60 kDa form of MT1-MMP bound gelatinase A at the cell surface. Both the activation of pro-MT1-MMP and the membrane binding of the tissue inhibitor of metalloproteinases type 2 (TIMP-2) and gelatinase A, and subsequent activation of gelatinase A, were inhi bited by calcium ionophores. Although the active MTI-MMP was required for cell surface binding and activation of gelatinase A, it was ineffi cient in activating gelatinase A in fibroblasts or in control MT-1080 cells alone. Low expression levels of TIMP-2 and rapid synthesis of MT I-MMP were found to be critical for gelatinase A activation. In MT-108 0 cells, MTI-MMP was further processed to an inactive, 43 kDa cell sur face form when overexpressed, or when the cells were treated with PMA, Under these conditions, the activated gelatinase A was detected in th e culture medium, in cell membrane extracts and in MT1-MMP-containing complexes. These results indicate that proteolytic processing (activat ion and degradation/ inactivation) of MTI-MMP and MT1-MMP/TIMP-2 relat ionships at the cell surface are important regulatory levels in the co ntrol of gelatinolytic activity.