Inhibition of matrix metalloproteinase 2 maturation and HT1080 invasiveness by a synthetic furin inhibitor

Abstract

The close correlation observed between matrix metalloproteinase 2 (MMP-2) activation and metastatic progression in various tumors suggests that MMP-2 is a`master switch' triggering tumor spread. Recently, membrane type 1 MMP (MT1-MMP) was identified as a potential physiological activator of MMP-2. Like all other MMPs, MT1-MMP possesses a pro-domain which must be removed for the enzyme to acquire its catalytic potential. The presence of a typical recognition motif (RXKR) for the furin-like convertases at the end of its pro-domain suggests a potential role for these proteinases in MT1-MMP processing. In order to evaluate the implication of furin in pro-MT1-MMP processing, we treated HT1080 cells with a synthetic furin inhibitor and monitored their ability to activate pro-MMP-2 as well as their invasive potential. Our results demonstrated that the furin inhibitor decreased pro-MT1-MMP processing as well as pro-MMP-2 activation and cell invasiveness. Therefore, our data bring further evidence that furin is a key factor in the maturation of MMPs associated with the invasive and metastatic potential of tumor cells.z 1998 Federation of European Biochemical Societies.

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

“…Analysis of gelatinolytic activities in conditioned media and total cell extracts was performed by gelatin zymography as previously described [27], The relative proportions of the gelatinolytic bands corresponding to the different forms of gelatinase A were evaluated by scanning densitometry using a GS-700 imaging densitometer (Bio-Rad, Hercules, CA) equipped with Molecular Analyst software. Results were expressed as relative percentages of total gelatinase A activity.…”

“…Samples of total cell extracts were analyzed using two distinct monoclonal antibodies (mAbs): mAb IE12 directed against the N-terminal prodomain of MT1-MMP and mAb 2D7 against the hemopexin-like domain of MT1-MMP (kindly provided by P. Basset, IGBMC, Illkirch, France) as previously described [27],…”

“…HT1080 cell chemoinvasion through type IV collagen (22 µg/insert) and chemotaxis were assayed using Transwell cell culture inserts (Costar) as previously described [27],…”

“…When HT1080 cells were plated on plastic, three major immunoreac-tive proteins were detected by Western blotting of total cell extracts: a doublet composed of 63-and 66-kDa species, both recognized by mAb 1E12 (Fig. 6A, lane 2) and corresponding to MT1-MMP proforms [27] as well as a 60-kDa band, revealed by mAb 2D7, corresponding to activated MT1-MMP (Fig. 6B, lane 2).…”

“…Sato and co-workers have identified MT1-MMP (membrane type 1 MMP or MMP-14): a MMP possessing a hydrophobic C-terminal transmembrane domain, which is considered as a physiological MMP-2 activator [22][23][24][25]. The active form of MT1-MMP, which is generated through a furin-dependent mechanism [26,27], binds TIMP-2 through the interaction of the N-terminal domains of each protein, thereby docking soluble TIMP-2 to the cell surface [20,[28][29][30]. This bimolecular complex subsequently functions as a receptor for the secreted pro-MMP-2, through interactions of their C-terminal domains, thus forming a MT1-MMP/TIMP-2/MMP-2 ternary complex.…”