Membrane-type Matrix Metalloproteinase-1 (MT1-MMP) Is a Processing Enzyme for Human Laminin γ2 Chain

Abstract

Processing of the laminin-5 (Ln-5) ␥2 chain by membrane-type-1 matrix metalloproteinases (MT1-MMP) promotes migration and invasion of epithelial and tumor cells. We previously demonstrated that MT1-MMP cleaves the rat ␥2 chain at two sites, producing two major C-terminal fragments of 100 (␥2) and 80 (␥2x) kDa and releasing a 30-kDa fragment containing epidermal growth factor (EGF)-like motifs (domain III (DIII) fragment). The DIII fragment bound the EGF receptor (EGF-R) and stimulated cell scattering and migration. However, it is not yet clear whether human Ln-5 is processed in a similar fashion to rat Ln-5 because one of the two MT1-MMP cleavage sites present in rat ␥2 is not found in human ␥2. To identify the exact cleavage site for MT1-MMP in human Ln-5, we purified both the whole molecule as well as a monomeric form of human ␥2 that is frequently expressed by malignant tumor cells. Like rat Ln-5, both the monomer of ␥2, as well as the ␥2 derived from intact Ln-5, were cleaved by MT1-MMP in vitro, generating C-terminal ␥2 (100 kDa) and ␥2x (85 kDa) fragments and releasing DIII fragments (25 and 27k Da). In addition to the conserved first cleavage site used to generate ␥2, two adjacent cleavage sites (Gly 559 -Asp 560 and Gly 579 -Ser 580 ) were found that could generate the ␥2x and DIII fragments. Two of the three EGF-like motifs present in the rat DIII fragment are present in the 27-kDa human fragment, and like the rat DIII, this fragment can promote breast carcinoma cell migration by engaging the EGF-R. These results suggest that MT1-MMP processing of Ln-5 in human tumors may stimulate the EGF-R, resulting in increased tumor cell scattering and migration that could possibly increase their metastatic potential.Laminin-5 (Ln-5), 1 a major component of the basement membrane, is a heterotrimer composed of ␣3, ␤3, and ␥2 subunits (1, 2). Migration and scattering of epithelial and tumor cells are induced by proteolytic processing of the ␥2 chain of Ln-5. The ␥2 chain is a 140-kDa polypeptide and forms a triple helix with the other subunits at its C-terminal (see Fig. 1A) (3, 4). Processing of the ␥2 chain occurs at the N terminus generating two major C-terminal fragments of 100 (␥2Ј) and 80 (␥2x) kDa (see Fig. 1A), and this processing has been observed in different species including humans and rodents (3, 4). Because of the limited availability of purified Ln-5, most biochemical studies in this area have been carried out using the rat protein. MT1-MMP and MMP-2 were identified as the proteases responsible for the second cleavage of the N terminus generating the ␥2x fragment (3, 4). In contrast, only MT1-MMP cleaved the first site to generate the ␥2Ј fragment (5). The two cleavage sites on the rat ␥2 chain were identified as Gly 434 -Asp 435 for ␥2Ј and Ala 586 -Leu 587 for ␥2x (3, 6). Processing of the rat ␥2 chain at these two sites releases an internal fragment containing three of the four EGF-like motifs in domain III (DIII) (7). Although Ln-5 does not stimulate the EGF receptor (EGF-R), the DIII fragment rele...

Laminin 5 Regulates Polycystic Kidney Cell Proliferation and Cyst Formation

“…MMP2 and MT1-MMP are enzymes known to induce ␥ 2 chain cleavage (25,26). Prolonged incubation of mature ␥ 2 chain with MMP2 generates 155-, 100-, 80-, 60-, and 30-kDa fragments (see Fig.…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…This process is thought to involve HD structural disassembly and to depend on proteolytic enzymes that remove the a3LG4-5 globules and proximal g2 short arm, promoting switching among stable anchorage and migration (Carter et al 1991;Rousselle et al 1991;Marinkovich et al 1992a;Marinkovich et al 1992b;Giannelli et al 1997;Koshikawa et al 2005). All of the laminins, with the exception of a1-laminins, undergo proteolytic cleavage of the LG domains and/or short arm.…”

Membrane-Type 1 Matrix Metalloproteinase Stimulates Cell Migration through Epidermal Growth Factor Receptor Transactivation

“…For example, EGFR is a major stimulus regulating MT1-MMP expression, as EGFR À/À mice show markedly decreased MT1-MMP expression (68). In addition, EGFR mediates the invasiveness of gliomas by inducing MT1-MMP (69) More recently, MT1-MMP -dependent proteolysis of the laminin-5 g 2 chain was shown to result in the formation of EGF ligands (70). As EGFR signaling is linked to diverse biological processes in human cancer cells, the identification of EGFR as a key component in the induction of MT1-MMP promigratory function implies a unique cooperation between pericellular proteolysis and intracellular signaling culminating in enhanced tumor growth and angiogenesis.…”