Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface

Abstract

Human fibroblasts and HT-1080 fibrosarcoma cells express membrane-type-1 matrix metalloproteinase (MT1-MMP), the cell surface activator of gelatinase A, in separate forms of 63 kDa, 60 kDa and in some cases 43 kDa. In the present work the interrelationships between MT1-MMP processing and gelatinase A activation were analysed using HT-1080 fibrosarcoma cells as a model. It was found that MT1-MMP was synthesized as a 63 kDa protein, which was constitutively processed to a 60 kDa active enzyme with N-terminal Tyr112, as shown by immunoprecipitation, immunoblotting and sequence analyses. Co-immunoprecipitation results indicated that only the active 60 kDa form of MT1-MMP bound gelatinase A at the cell surface. Both the activation of pro-MT1-MMP and the membrane binding of the tissue inhibitor of metalloproteinases type 2 (TIMP-2) and gelatinase A, and subsequent activation of gelatinase A, were inhibited by calcium ionophores. Although the active MT1-MMP was required for cell surface binding and activation of gelatinase A, it was inefficient in activating gelatinase A in fibroblasts or in control HT-1080 cells alone. Low expression levels of TIMP-2 and rapid synthesis of MT1-MMP were found to be critical for gelatinase A activation. In HT-1080 cells, MT1-MMP was further processed to an inactive, 43 kDa cell surface form when overexpressed, or when the cells were treated with PMA. Under these conditions, the activated gelatinase A was detected in the culture medium, in cell membrane extracts and in MT1-MMP-containing complexes. These results indicate that proteolytic processing (activation and degradation/inactivation) of MT1-MMP and MT1-MMP/TIMP-2 relationships at the cell surface are important regulatory levels in the control of gelatinolytic activity.

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

“…6B, lane 3). This latter MT1-MMP species was recently identified as a cell-surface-associated inactive form which results from an MMP-dependent processing of the 60-kDa form and whose occurrence was related to pro-MMP-2 activation [13,52]. In contrast to TPA, ConA did not increase the level of the 63-kDa pro-MT1-MMP but it dramatically increased both 60-and 43-kDa species (Figs.…”

Spectrum of matrix metalloproteinase expression in primary and metastatic colon cancer: relationship to the tissue inhibitors of metalloproteinases and membrane type-1-matrix metalloproteinase

“…However, it is the active form of MT-1-MMP and not the gene expression which is the key to MMP-2 activation. Lehti et al (1998) demonstrated that fibroblast MT-1-MMP was synthesized at a reduced rate compared to fibrosarcomas and is immediately complexed with membrane associated TIMP-2 and MMP-2, preventing activation, suggesting different post-translational regulation.…”

Regulation of Membrane Type-1 Matrix Metalloproteinase Activation by Proprotein Convertases

“…With the identification of the ϳ63-kDa species as the MT1-MMP zymogen, the ϳ60-and 45-kDa forms of MT1-MMP were noted to comigrate with those identified in HT-1080 cells ( Figure 2C, lanes 2 and 4), a cell line previously shown to process proMT1-MMP to both an ϳ60-kDa mature form and an ϳ45-kDa truncation product (Lohi et al, 1996;Lehti et al, 1998;Stanton et al, 1998). Consistent with this interpretation, a mAb directed at the catalytic domain of MT1-MMP identified the ϳ63-and 60-kDa forms of the enzyme, but not the 45-kDa fragment ( Figure 2C, lanes 5-8).…”

“…Cell monolayers were washed with ice-cold PBS (pH 8.0) and incubated with 0.5 mg/ml sulfo-NHS-biotin (Pierce) at 15°C for 40 min as described (Lehti et al, 1998). Cell lysates were incubated with strepavidin-Sepharose beads (Pierce) for 1 h at 4°C, and the complexes were resolved under reducing conditions by SDS-PAGE (10%).…”