The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1- Syndecans are transmembrane heparan sulfate proteoglycans expressed on all adherent cells (1, 2) and have been proposed to play an important role in tissue morphogenesis by virtue of their ability to bind, via their covalently attached glycosaminoglycan chains, to a variety of extracellular adhesive molecules including fibronectin, thrombospondin, various collagens, and heparin-binding growth-associated molecules and growth factors such as basic fibroblast growth factor (3-8).MMPSince the expression of syndecans appears to be controlled during both development and the progression of tumor cells to the metastatic phenotype, it has been proposed that syndecans are important regulators of the migratory and invasive behaviors of both normal and transformed cells (9, 10). The syndecan family is composed of four closely related proteins (syndecan-1, -2, -3, and -4) encoded by four different genes. Syndecan-1 is abundant in normal epithelial cells and tissues, localizing to both basal and suprabasal cell layers (1). Disruption of syndecan-1 expression in cultured cells leads to an epithelial mesenchymal transformation, with associated changes in cell polarity and cell-cell adhesion and altered epithelium-specific gene expression (7, 11).The intact ectodomain of each syndecan is constitutively shed from cultured cells (12, 13) as part of normal cell surface heparan sulfate proteoglycan turnover (14). Ectodomain shedding appears to contribute to diverse pathophysiological events such as host defense, wound healing, arthritis, and Alzheimer's disease, but how shedding is regulated remains largely unknown (15-17).Matrix metalloproteinases (MMPs) 1 are a family of Zn 2ϩ -dependent enzymes that are known to cleave extracellular matrix proteins in normal and pathological conditions (18 -20). To date, more than 20 mammalian MMPs have been identified by cDNA cloning, and they can be subgrouped into soluble type and membrane type MMPs (MT-MMPs) (20,21). MMPs are overexpressed in various human malignancies and have been thought to contribute to tumor invasion and metastasis by degrading extracellular matrix components (18,22). Thus, the level of MMP expression correlates with the invasiveness or malignancy of tumors (23,24). Particularly, MT1-MMP, MMP-2, MMP-7, and MMP-9 have been reported to be most closely associated with tumor invasion and metastasis. Whereas degradation of extracellular matrix is an important aspect of MMP biology, growing evidence has demonstrated specific processing/activation or degradation of cell surface receptors and ligands. Fas ligand (25), tumor necrosis factor-␣ (26), the ectodomain of the fibroblast growth factor receptor-1 (27), the heparin-binding epidermal growth factor (28), and interleukin-8 (29) were reported to be released or activated by MMPs. MMPs also cleave and inac...

Matrix metalloproteinases (MMPs) 1 are a family of Zn 2ϩ

Eph receptor tyrosine kinases and their ligands, ephrins, mediate neurodevelopmental processes such as boundary formation, axon guidance, vasculogenesis, and cell migration. We determined the expression profiles of the Eph family members in five glioma cell lines under migrating and nonmigrating conditions. EphB2 mRNA was overexpressed in all five during migration (1.2-2.8-fold). We found abundant EphB2 protein as well as strong phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2 localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc chimera stimulated migration and invasion of U87, whereas treatment with a blocking EphB2 antibody significantly inhibited migration and invasion. Forced expression of EphB2 in U251 cells stimulated cell migration and invasion and diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251 stably transfected with EphB2 showed more scattered and more pronounced invasive growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2 expression was higher in glioblastomas than in lowgrade astrocytomas or normal brain; patterns of phosphorylated EphB2 matched the expression levels. Laser capture microdissection of invading glioblastoma cells revealed elevated EphB2 mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases) and not in normal brain. This is the first demonstration that migrating glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. Dysregulation of EphB2 expression or function may underlie glioma invasion.

Degradation of extracellular matrix (ECM) is(1-3) Under normal physiological conditions, the activities of MMP are precisely regulated at the level of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous inhibitors.(4) Cancer cells also make use of MMP for invasion and metastasis. The invading cells are forced to proliferate within an embedded dense three-dimensional matrix composed largely of type I collagen or cross-linked fibrin. Membrane type MMP-1 (MT1-MMP) has been thought to play a major role in this step. (5,6) Unique structure of MT1-MMP. Currently 23 MMP have been identified in humans and they are divided into two groups; secretory and membrane-type (Fig. 1).(7) While MT1-MMP has a common MMP domain structure with pre-, pro-, catalytic and hemopexin-like domains, it also has unique insertions. First there is an insertion of 11 amino-acids between the pro-peptide and the catalytic domains, which contains the recognition sequence for the Golgi-associated processing enzyme furin. Another unique insertion at the C-terminus contains a hydrophobic amino-acid sequence that acts as a transmembrane domain. Thus, unlike secretory MMP, MT1-MMP is expressed on the cell surface in an active form. Implication of MT1-MMP in tumor invasion and metastasis. MMP-2 has been suspected to be associated with tumor invasion because it can degrade the type IV collagen, which is a major component of the basement membrane. An active form of MMP-2 has frequently been detected in tumor tissues. (8,9) The expression of MT1-MMP has been demonstrated in tumor tissues, in which MMP-2 is always activated.(5,10 -12) Furthermore, the MMP-2 activation ratio correlates with the MT1-MMP expression level, suggesting that MT1-MMP is the MMP-2 activator in tumor tissues. An analysis of MT1-MMP-deficient mice, which had severe defects in bone formation and died within a few weeks of birth, clearly showed that MT1-MMP is the major activator of MMP-2 during tissue morphogenesis.(13) Interestingly MMP-2-deficient mice do not show such severe defects, indicating that MT1-MMP has critical functions in addition to MMP-2 activation. ECM macromolecules including type I collagen and various biologically important molecules are now known to serve as substrates for MT1-MMP, which will be discussed in the following sections (Table 1). (14 -23) Induction of MT1-MMP expression by transformation of epithelial cells. MT1-MMP is associated with not only tumor invasion and metastasis but also tissue morphogenesis, which is clearly demonstrated in vitro by the Madin-Darby canine kidney (MDCK) epithelial cell system (Fig. 2). MDCK cells are kidney-derived normal epithelial cells that show typical cobblestone morphology when cultured in a plastic dish. MDCK cells form cysts when cultured in three-dimensional type I collagen gel, and grow to form branching tubules in the presence of hepatocyte growth factor.(24) MT1-MMP expression is induced at a low level in tube-forming MDCK cells. Collageno...

A human placenta cDNA library was screened by the expression cloning method for gene products that interact with matrix metalloproteinases (MMPs), and we isolated a cDNA whose product formed a stable complex with pro-MMP-2 and pro-MMP-9. The cDNA encoded the metastasis suppressor gene KiSS-1. KiSS-1 protein was shown to form a complex with pro-MMP. KiSS-1 protein is known to be processed to peptide ligand of a G-proteincoupled receptor (hOT7T175) named metastin, and suppresses metastasis of tumors expressing the receptor. Active MMP-2, MMP-9, MT1-MMP, MT3-MMP and MT5-MMP cleaved the Gly 118 -Leu 119 peptide bond of not only full-length KiSS-1 protein but also metastin decapeptide. Metastin decapeptide induced formation of focal adhesion and actin stress fibers in cells expressing the receptor, and digestion of metastin decapeptide by MMP abolished its ligand activity. Migration of HT1080 cells expressing hOT7T175 that harbor a high-level MMP activity was only slightly suppressed by either metastin decapeptide or MMP inhibitor BB-94 alone, but the combination of metastin decapeptide and BB-94 showed a synergistic effect in blocking cell migration. We propose that metastin could be used as an antimetastatic agent in combination with MMP inhibitor, or MMP-resistant forms of metastin could be developed and may also be efficacious.