TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP.
The matrix metalloproteinase (MMP)-2 has a crucial role in extracellular matrix degradation associated with cancer metastasis and angiogenesis. The latent form, pro-MMP-2, is activated on the cell surface by the membrane-tethered membrane type 1 (MT1)-MMP, in a process regulated by the tissue inhibitor of metalloproteinase (TIMP)-2. A complex of active MT1-MMP and TIMP-2 binds pro-MMP-2 forming a ternary complex, which permits pro-MMP-2 activation by a TIMP-2-free neighbouring MT1-MMP. It remains unclear how MMP-2 activity in the pericellular space is regulated in the presence of TIMP-2. To address this question, the effect of TIMP-2 on MMP-2 activity in the extracellular space was investigated in live cells, and their isolated plasma membrane fractions, engineered to control the relative levels of MT1-MMP and TIMP-2 expression. We show that both free and inhibited MMP-2 is detected in the medium, and that the net MMP-2 activity correlates with the level of TIMP-2 expression. Studies to displace MT1-MMP-bound TIMP-2 in a purified system with active MMP-2 show minimal displacement of inhibitor, under the experimental conditions, due to the high affinity interaction between TIMP-2 and MT1-MMP. Thus inhibition of MMP-2 activity in the extracellular space is unlikely to result solely as a result of TIMP-2 dissociation from its complex with MT1-MMP. Consistently, immunoblot analyses of plasma membranes, and surface biotinylation experiments show that the level of surface association of TIMP-2 is independent of MT1-MMP expression. Thus low-affinity binding of TIMP-2 to sites distinct to MT1-MMP may have a role in regulating MMP-2 activity in the extracellular space generated by the ternary complex.
Bernardo
M Margarida
MM
Department of Pathology, School of Medicine, Wayne State University, 540 E. Canfield Avenue, Detroit, MI 48201, USA.
Fridman
Rafael
R
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004793:Enzyme Reactivators / Q000378:metabolism; D004847:Epithelial Cells / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D005109:Extracellular Matrix / Q000201:enzymology*; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism* / Q000502:physiology*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D004793:Enzyme Reactivators; D019716:Tissue Inhibitor of Metalloproteinase-2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2