Kazushi Imai

TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads

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.

Cell surface binding and activation of gelatinase A induced by expression of membrane-type-1-matrix metalloproteinase (MT1-MMP)

Gelatinase A is secreted as a proenzyme (progelatinase A) which is activated and bound on the surface of tumor and normal cells. We have reported that the expression of a membrane‐type‐1‐matrix metalloproteinase (MT1‐MMP) induces activation of progelatinase A. Here we demonstrate that the expression of MT1‐MMP in COS‐1 cells induces cell‐surface binding of progelatinase A which is consequently processed to an intermediate form. Processing from the intermediate to the fully active form is dependent on the gelatinase A concentration. These results suggest that the cell‐surface binding concentrates the gelatinase A intermediate form locally to allow autoproteolytic processing to the fully active form.

Susceptibility of tenascin to degradation by matrix metalloproteinases and serine proteinases

The degradation of tenascin purified from human melanoma cells was examined by treatment with matrix metalloproteinases (MMPs) and serine proteinases. Among eight different types of proteinases examined, MMP‐1, ‐3, and ‐7, cathepsin G and leukocyte elastase could digest tenascin, but MMP‐2, MMP‐9 and thrombin did not. This suggests that tenascin may be readily catabolized by extracellular matrix‐degrading proteinases found in the pathophysiological conditions.

Degradation of vitronectin by matrix metalloproteinases-1, -2, -3, -7 and -9

The susceptibility of vitronectin (Vn) purified from human plasma to digestion by matrix metalloproteinases (MMPs) was examined. MMP‐2, ‐3, ‐7 and ‐9 except for MMP‐1 degraded Vn into multiple fragments. MMP‐7 showed the highest activity to the substrate among these MMPs, digesting 8‐, 30‐ and 44‐fold more preferentially than MMP‐2, ‐3 and ‐9, respectively. These data suggest that MMP‐2, ‐3, ‐7 and ‐9 may be responsible for the pathological degradation and/or normal turnover of Vn.

Complement Cls, a classical enzyme with novel functions at the endochondral ossification center: immunohistochemical staining of activated Cls with a neoantigen-specific antibody

Abstract.The secondary ossification center of 14- to 16-day-old hamster tibiae was examined immunohistochemically with active and inactive Cls-specific antibodies, RK5 and RK4, respectively. At the ossification center, chondrocytes differentiate from proliferating and hypertrophic to degenerating stages, and their site is occupied by the bone marrow. Cls was strongly immunostained in hypertrophic chondrocytes. In order to discover whether Cls is activated at a particular site, the cartilage was immunostained with RK5 and RK4. RK5 mainly reacted with degrading matrix around invading vessels. In contrast, RK4 strongly stained hypertrophic chondrocytes. Immunoelectron microscopy revealed Cls on degrading fragments of chondrocytes and fibers of cartilage matrix. Decorin, one of the major matrix proteoglycans, was dose and time dependently degraded by Cls. Type II collagen and type I gelatin were also degraded. Articular cartilage from patients with rheumatoid arthritis was positively immunostained (11/12 cases) with an anti-Cls monoclonal antibody (mAb) PG11, whereas normal articular cartilage (5/5 cases) was negative, suggesting Cls participation in the etiology of rheumatoid arthritis.

Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of αv integrins

We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3, poorly differentiated type; OSC-19, well-differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-αv antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-β1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-β5 and anti-αv antibodies, and only HSC-3 cells were positive for anti-β3 antibody. α5β1 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects αvβ3-and/or αvβ5-mediated carcinoma cell invasion.