Tissue Inhibitor of Metalloproteinase (TIMP)-2 Acts Synergistically with Synthetic Matrix Metalloproteinase (MMP) Inhibitors but Not with TIMP-4 to Enhance the (Membrane Type 1)-MMP-dependent Activation of Pro-MMP-2

Marta Toth,M Margarida Bernardo,David C Gervasi,Paul D Soloway,Zhiping Wang,Heather F Bigg,Christopher M Overall,Yves A Declerck,Harald Tschesche,Michael L Cher,Stephen Brown,Shahriar Mobashery,Rafael Fridman

doi:10.1074/jbc.m006871200

Abstract

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.

Highlights

Proteolytic degradation of extracellular matrix (ECM)1 is a fundamental aspect of cancer development and a key event in tumor-induced angiogenesis and tumor metastasis
We show for the first time that synthetic MMP inhibitors (MMPIs) and a C-terminally truncated tissue inhibitors of metalloproteinases (TIMPs)-2 but not TIMP-4, which inhibit MT1-matrix metalloproteinase (MMP) activity, act synergistically with TIMP-2 to promote pro-MMP-2 activation by MT1-MMP
The cells were tested for activation of exogenous pro-MMP-2 after treatment with concanavalin A [66], and neither cell variant activated pro-MMP-2 regardless of the presence of TIMP-2, suggesting a low level of endogenous MT1-MMP expression

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Nonmalignant monkey kidney epithelial BS-C-1 (CCL26) and human fibrosarcoma HT-1080 (CCL-121) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. Natural and Synthetic Inhibitor Treatment and Pro-MMP-2 Activation—After infection, the media were aspirated, and the cells were rinsed with serum-free DMEM and replaced with fresh serum-free DMEM supplemented with or without various doses of purified human recombinant TIMP-2. Progress curves were obtained by adding enzyme (0.5 nM) to a mixture of fluorogenic substrate (7 ␮M) and varying concentrations of inhibitor in buffer R (50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 0.01% Brij-35, and 1–5% Me2SO; final volume 2 ml) in acrylic cuvettes with stirring and monitoring the increase in fluorescence with time for 15–30 min. Initial rates were obtained by adding enzyme (0.5 nM) to a mixture of fluorogenic substrate (7 ␮M) and varying concentrations of inhibitor in buffer R (final volume 1 ml) in quartz semimicro cuvettes and monitoring the increase in fluorescence with time for 5–10 min. Where vi and v0 represent the initial velocity in the presence and absence of inhibitor, respectively, using the program SCIENTIST

RESULTS

TABLE I

Inhibitor kon koff

DISCUSSION