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

Biochem J. 2003 Sep 15;374(Pt 3):739-45. doi: 10.1042/BJ20030557.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Fractionation
  • Cell Line
  • Cell Membrane / enzymology
  • Enzyme Activation
  • Enzyme Precursors / metabolism*
  • Enzyme Reactivators / metabolism
  • Epithelial Cells / chemistry
  • Epithelial Cells / enzymology
  • Epithelial Cells / metabolism
  • Extracellular Matrix / enzymology*
  • Haplorhini
  • HeLa Cells
  • Humans
  • Kidney / cytology
  • Matrix Metalloproteinase 2 / metabolism*
  • Matrix Metalloproteinase 2 / physiology*
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases / biosynthesis
  • Metalloendopeptidases / metabolism*
  • Tissue Inhibitor of Metalloproteinase-2 / metabolism
  • Tissue Inhibitor of Metalloproteinase-2 / physiology*
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Enzyme Precursors
  • Enzyme Reactivators
  • Tissue Inhibitor of Metalloproteinase-2
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases
  • Matrix Metalloproteinase 2