Membrane Type 1 Matrix Metalloproteinase-associated Degradation of Tissue Inhibitor of Metalloproteinase 2 in Human Tumor Cell Lines

Abstract

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been shown to function as a receptor for secreted pro-MMP-2, resulting in the formation of a trimolecular complex. In the presence of uncomplexed active MT1-MMP, the prodomain of cell surface-associated MMP-2 is cleaved, and activated MMP-2 is released. However, the behavior of MT1-MMP-bound TIMP-2 during MMP-2 activation is currently unknown. In this study, 125 I-labeled recombinant TIMP-2 ( 125 I-rTIMP-2) was used to investigate the fate of TIMP-2 during pro-MMP-2 activation by HT1080 and transfected A2058 cells. HT1080 and A2058 cells transfected with MT1-MMP cDNA (but not vector-transfected A2058 cells) were able to bind 125 I-rTIMP-2, to activate pro-MMP-2, and to process MT1-MMP into an inactive 43-kDa form. Under these conditions, 125 IrTIMP-2 bound to the cell surface was rapidly internalized and degraded in intracellular organelles through a bafilomycin A 1 -sensitive mechanism, and 125 I-bearing low molecular mass fragment(s) were released in the culture medium. These different processes were inhibited by hydroxamic acid-based synthetic MMP inhibitors and rTIMP-2, but not by rTIMP-1 or cysteine, serine, or aspartic proteinase inhibitors. These results support the concept that the MT1-MMP-dependent internalization and degradation of TIMP-2 by some tumor cells might be involved in the regulation of pericellular proteolysis.

Type IV Collagen Induces Matrix Metalloproteinase 2 Activation in HT1080 Fibrosarcoma Cells

“…9A). We have previously demonstrated that this low-molecular-weight band represents 125 I-bearing peptide (s) resulting from the degradation of 125 I-TIMP-2 [54]. Quantification of degraded 125 I-rTIMP-2 by scanning densitometry of the autoradiographs revealed that HT1080 cells plated on type IV collagen degraded 2x more 125 I-rTIMP-2 than those plated on plastic (Fig.…”

“…In a previous study, we demonstrated that TPA treatment of HT1080 cells stimulated the internalization and degradation of TIMP-2 through a MT1-MMP-dependent mechanism, resulting in a lower level of secreted TIMP-2 [54]. To investigate the potential occurrence of a similar degradation in the presence of type IV collagen, we compared the fate of 125 I-rTIMP-2 (0.6 nM) added to HT1080 cells cultured for up to 48 h on plastic or type IV collagen (20 µg/well).…”

“…7D). We recently demonstrated that this low concentration of secreted TIMP-2 resulted from an MT1-MMP-dependent internalization of TIMP-2 associated with an intracellular degradation process [54]. When HT1080 cells were plated on type IV collagen, the cell-associated TIMP-2 concentration was slightly increased (Figs.…”

“…Such synthetic inhibitors have previously been shown to bind to the active site of MMPs [55], thus competing with TIMP-2 for binding to MT1-MMP [28,33,54]. HT1080 cells plated on type IV collagen were incubated for up to 48 h with or without 1 µM GI129471 and TIMP-2 concentrations in conditioned media and total cell extracts were determined by ELISA (Fig.…”

“…On the basis of the dual role of TIMP-2 during MMP-2 activation, promoting MMP-2 activation when present at low concentrations [20,29,31,54,63,64], but acting as an efficient inhibitor of this process when present at higher doses [19,20,26,31,53,[65][66][67], we hypothesized that modulations of TIMP-2 concentration could play important roles in the regulation of MMP-2 processing. Consequently, we investigated the modifications of both TIMP-2 concentration and distribution between conditioned media and cell extracts induced by MMP-2-activating treatments.…”