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Membrane-type matrix metalloproteinases (MT-MMPs) have emerged as key enzymes in tumor cell biology. The importance of MT1-MMP, in particular, is highlighted by its ability to activate pro-MMP-2 at the cell surface through the formation of a trimolecular complex comprised of MT1-MMP/tissue inhibitor of metalloproteinase-2 (TIMP-2)/pro-MMP-2. TIMPs 1-4 are physiological MMP inhibitors with distinct roles in the regulation of pro-MMP-2 processing. Here, we have shown that individual Timp deficiencies differentially affect MMP-2 processing using primary mouse embryonic fibroblasts (MEFs). Timp-3 deficiency accelerated pro-MMP-2 activation in response to both cytochalasin D and concanavalin A. Exogenous TIMP-2 and N-TIMP-3 inhibited this activation, whereas TIMP-3 containing matrix from wild-type MEFs did not rescue the enhanced MMP-2 activation in Timp-3(-/-) cells. Increased processing of MMP-2 did not arise from increased expression of MT1-MMP, MT2-MMP, or MT3-MMP or altered expression of TIMP-2 and MMP-2. To test whether increased MMP-2 processing in Timp-3(-/-) MEFs is dependent on TIMP-2, double deficient Timp-2(-/-)/-3(-/-) MEFs were used. In these double deficient cells, the cleavage of pro-MMP-2 to its intermediate form was substantially increased, but the subsequent cleavage of intermediate-MMP-2 to fully active form, although absent in Timp-2(-/-) MEFs, was detectable with combined Timp-2(-/-)/-3(-/-) deficiency. TIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.

Original publication

DOI

10.1074/jbc.m512009200

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

04/2006

Volume

281

Pages

10337 - 10346

Addresses

Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada.

Keywords

Cell Line, Fibroblasts, Animals, Mice, Inbred C57BL, Mice, Transgenic, Humans, Mice, Cytochalasin D, Matrix Metalloproteinases, Proteins, Concanavalin A, Recombinant Proteins, Tissue Inhibitor of Metalloproteinases, Tissue Inhibitor of Metalloproteinase-2, Tissue Inhibitor of Metalloproteinase-3, RNA, Messenger, Blotting, Western, Electrophoresis, Polyacrylamide Gel, Crosses, Genetic, Reverse Transcriptase Polymerase Chain Reaction, Enzyme Activation, Protein Binding, Models, Biological, Time Factors, Cricetinae, Matrix Metalloproteinases, Membrane-Associated, Matrix Metalloproteinase 2, Matrix Metalloproteinase 14, Matrix Metalloproteinase 15, Matrix Metalloproteinase 16, Embryo, Mammalian