Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.
Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP. In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2. The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner. In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations. TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA. A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available. Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F. Matrix metalloproteinases (MMPs)1 are zinc-dependent endopeptidases that play critical roles in the physiological and pathological turnover of extracellular matrix (ECM) by degrading the macromolecules (1-6). MMPs are produced as a zymogen (proMMP) that needs proteolytic activation by eliminating the N-terminal propeptide for the enzymes to function (7). Serine proteases such as plasmin, neutrophil elastase, and trypsin are well known activators for proMMPs. These activators digest the propeptide sequences at the basic amino acid motifs and eventually induce autocatalytic activation (8).However, proGelA lacks such a basic motif and therefore cannot be activated by serine proteinases (9). ProGelA had been reported to be activated by an unknown MMP-like activity on the surface of cancer and fibroblastic cells (10 -15), and we identified MT1-MMP as such an activator on the cell surface (16,17). Three other genes encoding similar enzymes that have a transmembrane domain and a short cytoplasmic tail were identified (18 -20); at least two of them (MT2-MMP and MT3-MMP) activated proGelA in vitro (21).Upon cell-mediated activation, proGelA binds to the cells through its hemopexin-like domain (HLD) (22). Using the HLD of GelA, Strongin et al. (23) isolated TIMP-2 complexed with the activated form of MT1-MMP from the cell membrane extract. We also purified a shaded fragment of MT1-MMP from the culture medium of the human breast carcinoma cell line MDA-MB-231 as a form inhibited by TIMP-2 (24). The Cterminal domain of the TIMP-2 in the complex was available for further complex formation with proGelA through its HLD (trimolecular complex). Strongin et al. also demonstrated that a small amount of TIMP-2 is an essential component for the activation of proGelA on the surface, in contrast to evidence that TIMP-2 is a well established inhibitor for all of the known MMPs. It would thus be of inter...
ADAMs (a disintegrin and metalloproteinases) are multifunctional molecules involved in cell-cell fusion, cell adhesion, membrane protein shedding, and proteolysis. In the present study, we examined the mRNA expression of 13 different ADAM species with putative metalloproteinase activity in human astrocytic tumors, nonneoplastic brain tissues, and other intracranial tumors by reverse transcriptase-polymerase chain reaction, and found that prototype membrane-anchored ADAM12 (ADAM12m) is predominantly expressed in glioblastomas. Real-time quantitative polymerase chain reaction indicated that the expression level of ADAM12m is remarkably at least 5.7-fold higher in glioblastomas (n ؍ 16) than in nonneoplastic brain tissues (n ؍ 6), low grade (n ؍ 7) and anaplastic astrocytic tumors (n ؍ 9) (P < 0.05 for each group), and intracranial neurinomas (n ؍ 5) (P < 0.01). In situ hybridization showed that glioblastoma cells are responsible for the gene expression. ADAMs (a disintegrin and metalloproteinases) are a gene family of multidomain membrane-anchored proteins comprising of more than 30 members in various animal species (see http://www.people.virginia.edu/ϳjw7g/Tableof theADAMs.html) and are implicated in pathophysiological conditions, which include neuronal development, 1 cancer development and progression, 2,3 and inflammatory responses 4 through proteolysis, cell adhesion, cell fusion, and cell-matrix interaction. 5,6 They contain several distinct domains with structural homology to the reprolysin/adamalysin family of snake venom metalloproteinases.7 A typical ADAM protein includes an N-terminal signal peptide, and propeptide, metalloproteinase, disintegrin, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains. The metalloproteinase domains of several ADAMs have a catalytic site with the conventional zinc-dependent metalloproteinase sequence (HEXGHXXGXXHD), which is highly homologous to that of the matrix metalloproteinases (MMPs).
ADAM (a disintegrin and metalloproteinases) are a recently discovered gene family of proteins with sequence similarity to the reprolysin family of snake venom metalloproteinases, and about one-third of the family members have the catalytic site consensus sequence in their metalloproteinase domains. We screened the mRNA expression of 11 different ADAM species with putative metalloproteinase activity in human non-small cell lung carcinomas by RT-PCR, and found that prototype membrane-anchored ADAM28 (ADAM28m) and secreted ADAM28 (ADAM28s) are predominantly expressed in the carcinoma tissues. Real-time quantitative PCR demonstrated that the expression levels of ADAM28m and ADAM28s are significantly 16.8-fold and 9.0-fold higher in the carcinomas than in the non-carcinoma tissues, respectively. In addition, the expression levels of ADAM28m and ADAM28s were significantly higher in the carcinomas with >30 mm in diameter than in those 530 mm. The expression levels were also significantly higher in the carcinomas with lymph node metastasis than in those without metastasis. MIB1-positive cell index of the carcinomas had a direct correlation with the expression levels of ADAM28m and ADAM28s (r 5 0.667, p < 0.001 and r 5 0.535, p < 0.01, respectively). In situ hybridization and immunohistochemistry demonstrated that ADAM28 is expressed predominantly in the carcinoma cells. Immunoblot analysis showed the activated form of ADAM28 in the carcinoma tissues. These data demonstrate for the first time that ADAM28 is overexpressed and activated in human non-small cell lung carcinomas, and suggest the possibility that ADAM28 plays a role in cell proliferation and progression of the human lung carcinomas. ' 2005 Wiley-Liss, Inc.Key words: ADAM; MMP; lung cancer; proliferation; metastasis ADAM (a disintegrin and metalloproteinases) are a gene family that have significant sequence similarity to the reprolysin/adamalysin family of snake venom metalloproteinases.1 ADAM are composed of several domains including propeptide, metalloproteinase, disintegrin, cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and cytoplasmic tail domains. More than 30 members of the ADAM gene family have been identified in a variety of animal species (see http://www.people.virginia.edu/ jw7g/ Table_of_the_ADAMs.html). Although the specific biological functions of ADAM are not clear, they may be involved in shedding of various membrane-anchored receptors and proteins, degradation of extracellular matrix (ECM), and cell adhesion and migration.2,3 About one-third of the ADAM members have the catalytic consensus sequence (HEXGHXXGXXHD) in their metalloproteinase domains, and are predicted to have catalytic activity. ADAM10 is reported to digest myelin basic protein 4 and type IV collagen.5 ADAM9 degrades insulin B chain, 6 and ADAM28 cleaves myelin basic protein 7 and insulin-like growth factor binding protein-3 (IGFBP-3). 8 In addition, the precursor of tumor necrosis factor-a (proTNF-a) is processed to the mature form by ADAM10 and ADAM17. 9 CD23 ...
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