Paradigmatic identification of MMP‐2 and MT1‐MMP activation systems in cardiac fibroblasts cultured as a monolayer

Abstract

Activations of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin-dependent MMP-2 and MT1-MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP-2 and MT1-MMP activations by potential non-matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype-producing agent. We showed that treatment of cells with ConA activated pro-MMP-2, and that this activation concurred with elevated levels of cellular MT1-MMP and TIMP-2. The presence of active MT1-MMP and 43 and 36 kDa processed forms of MT1-MMP in a fraction of intracellular proteins prepared from ConA-treated cells suggests the possible internalization of differential forms of MT1-MMP. The appearance of 36 kDa processed form of MT1-MMP in conditioned media prepared from ConA-treated cells indicates the possible extracellular release of the further processed MT1-MMP fragment. Inhibition of furin in ConA-treated cells attenuated pro-MT1-MMP processing and the cellular TIMP-2 level, plus it reduced cell-released active MMP-2 in a time-dependent manner. These results suggest the involvement of furin in the ConA-induced activations of MT1-MMP and MMP-2. Furthermore, the existence of furin inhibitor-insensitive pro- and active MMP-2 species associated with ConA-treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP-2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif.

Regulation of MT1-MMP and MMP-2 by Leptin in Cardiac Fibroblasts Involves Rho/ROCK-Dependent Actin Cytoskeletal Reorganization and Leads to Enhanced Cell Migration

“…Our data also demonstrate that Rho/ROCK signaling and subsequent cytoskeletal rearrangement mediated increased localization of MT1-MMP to the plasma membrane in response to leptin, which may have several important functional consequences (9,26,38,39). First, based on our previous work in cardiac fibroblasts (9), we further investigated whether leptin-induced increased cell surface localization of MT1-MMP regulated activation of secreted pro-MMP-2.…”

Mutational and Structural Analyses of the Hinge Region of Membrane Type 1-Matrix Metalloproteinase and Enzyme Processing

“…We found that although arginine substitutions at the Gly 284 -Gly 285 site inhibited formation of the 44-kDa species, they did not prevent processing. Instead, the MT1-MMP mutants underwent alternate cleavages at downstream sites, within the hinge region, to generate smaller degradation products (ϳ41-kDa), similar to those detected under conditions of enzyme overexpression (31, 36 -38) or inhibition of endocytosis (39). The pattern of processing exhibited by a series of MT1-MMP C-terminal hinge deletion mutants, which preserve the Gly 284 -Gly 285 site, points to the region located between Gln 296 and Ser 304 of the hinge region as the main target for the additional cleavage site(s) during processing.…”

“…The processing of MT1-MMP is a cell surface event in which the active enzyme is autocatalytically cleaved in trans to a major 44-kDa membrane-tethered degradation form (also referred to as the 43-or 45-kDa species) lacking the catalytic domain. A series of minor degradation products of 43-40 kDa can also be detected under conditions of enzyme overexpression (31, 36 -38) or exposure of cells to concanavalin A, which inhibits MT1-MMP endocytosis (39). Concomitantly, a fragment of the catalytic domain is released into the extracellular space as an inactive 18 -20-kDa soluble form (30,33).…”

“…Extracts of primary human glioma invasive tissues contained active and processed MT1-MMP (47). In cultured cells, the extent of MT1-MMP processing is influenced by factors that regulate enzyme expression, clustering, endocytosis, and/or lysosomal degradation such as phorbol ester (29,(51)(52)(53), concanavalin A (33,39,51,54,55), bafilomycin A1 (55,56), calmodulin inhibitors (57), and calcium ionophores (29,58). Thus, processing of active MT1-MMP to the 44-kDa species is a physiological mechanism of enzyme turnover.…”

Proprotein convertases: “Master switches” in the regulation of tumor growth and progression

“…This concept has been supported by Sato et al, who demonstrated that recombinant MT1‐MMP was efficiently cleaved in vitro by purified soluble furin [27], [42,45,46]. Interestingly, furin inhibition either with a synthetic inhibitor (decanoyl‐Arg‐Val‐Lys‐Arg‐chloromethylketone, or CMK) or antisense technology resulted in a reduced Pro‐MT1‐MMP and MMP‐2 activitation in cardiac and uterine cervical fibroblasts [47,48] but not in rabbit dermal fibroblasts [48]. These results suggest that furin involvement in MT1‐MMP maturation and consequently in the regulation of collagenolytic modulation may be species‐, tissue‐, and/or cell type‐dependent.…”