Distinct Roles for the Catalytic and Hemopexin Domains of Membrane Type 1-Matrix Metalloproteinase in Substrate Degradation and Cell Migration

Abstract

Substrate degradation and cell migration are key steps in cancer metastasis. Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Using the fluorescein isothiocyanate (FITC)-labeled fibronectin degradation assay combined with the phagokinetic cell migration assay, structurefunction relationships of MT1-MMP were studied. Our data indicate that MT1-MMP initiates substrate degradation and enhances cell migration; cell migration occurs as a concurrent but independent event. Using recombinant DNA approaches, we demonstrated that the hemopexin-like domain and a nonenzymatic component of the catalytic domain of MT1-MMP are essential for MT1-MMP-mediated cell migration. Because the cytoplasmic domain of MT1-MMP was not required for MT1-MMP-mediated fibronectin degradation and cell migration, it is proposed that cross-talk between the hemopexin domain of MT1-MMP and adjacent cell surface molecules is responsible for outside-in signaling. Employing cDNAs encoding dominant negative mutations, we demonstrated that Rac1 participates in the MT1-MMP signal transduction pathway. These data demonstrated that each domain of MT1-MMP plays a distinct role in substrate degradation and cell migration.Cell migration and invasion are critical coordinated events in the cancer dissemination process (1, 2). Cell migration involves the locomotion of a cell over an extracellular matrix (ECM) 1 substratum (3). Extension of the leading edge is associated with adhesion, i.e. binding of integrins to their ECM ligands leading to subsequent migration and further invasion (4). Cancer cell invasion requires degradation of surrounding ECM and basement membrane by proteinases located at the leading edge of migrating cells. Extracellular proteolytic enzymes, i.e. matrix metalloproteinases (MMPs), serine and cysteine proteinases have long been implicated in cancer metastasis (1, 5).MMPs have been linked to the metastatic phenotype of tumor cells through both correlative and functional studies. Production and activation of MMPs in tumors are required for degradation of the ECM and dissemination of cancer cells to distant organs (2). MMPs also play an important role in tumor angiogenesis (6). The mechanism of activation of latent MMP-2 (pMMP-2) in tumors has been the focus of considerable recent interest based on the identification of a new category of intrinsic membrane-type MMPs (MT1, 2, 3, 4, 5, and 6-MMPs) (7).MT1-MMP is able to activate pMMP-2 on the surface of tumor cells by assembling a unique triplex with tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and pMMP-2; a second MT1-MMP molecule then cleaves the propeptide of pMMP-2, thereby activating the enzyme at the cell surface (8 -10). Integrin receptors, ␣ 3 ␤ 1 and ␣ v ␤ 3 , participate in this response (11). Recombinant MT1-MMP hydrolyze collagen types I, II, and III and digests cartilage proteoglycan, fibronectin, fibrinogen, vitronectin, and laminin (12).It is now recognized that the actions of MMPs are not restricted to the simple breakdown of ECM...

Gelatinase-mediated migration and invasion of cancer cells

“…It has become clear that protease-independent migration strategies exist. Recently it was shown that catalytically inactive MT1-MMP mutant supported cell migration similarly to the wild type enzyme and the cell migration supporting activity was accounted for the catalytic domain and the Cterminal domain (Cao et al, 2004). Other investigators have similarly suggested a migration inducing ability of MMP-9 independent of the catalytic activity (Sanceau et al, 2003).…”

“…Another point to note is that the proteinases have functions independent on their proteolytic activity. This has been well established with the uPA/uPAR system (Blasi and Carmeliet, 2002) and evidence is accumulating that the MMPs function similarily (Cao et al, 2004;Sanceau et al, 2003).…”

MT1-MMP is the critical determinant of matrix degradation and invasion by ovarian cancer cells

“…Several studies have reported motility to be somewhat enhanced by overexpression of wild-type MT1 (Gingras et al, 2001;Rozanov et al, 2001;Cao et al, 2004;Takino et al, 2004). MT1 effects on cell motility may be exerted through integrin activation, as MT1-mediated cleavage of the vitronectin receptor avb3 enhanced breast cancer cell motility on this substrate (Deryugina et al, 2002).…”

“…To determine whether MT1 also promoted the migration of ovarian cancer cells, as demonstrated in other cancer cell types (Gingras et al, 2001;Rozanov et al, 2001;Cao et al, 2004;Takino et al, 2004), the transfected cells were subjected to a transwell collagen migration assay. In contrast to its striking effect on collagen degradation, MT1 expression did not enhance cell migration ( Figure 4B).…”

Membrane type-matrix metalloproteinases and tumor progression

“…More recently, Takino et al [107] demonstrate that MT1-MMP-mediated MEK/ERK activation leads to tumor cell invasion in type-1 collagen gel. By using cDNAs encoding dominant negative inhibitors targeted to different signaling pathways, Rac 1 activation by MT1-MMP has also been involved in cell migration [108]. Processing of integrin α v subunit by MT1-MMP leads to tyrosine phosphorylation of focal adhesion kinase and cell migration [100].…”