ADAMTS10 protein interacts with fibrillin-1 and promotes its deposition in extracellular matrix of cultured fibroblasts.

Autosomal recessive and autosomal dominant forms of Weill-Marchesani syndrome, an inherited connective tissue disorder, are caused by mutations in ADAMTS10 (encoding a secreted metalloprotease) and FBN1 (encoding fibrillin-1, which forms tissue microfibrils), respectively, yet they are clinically indistinguishable. This genetic connection prompted investigation of a potential functional relationship between ADAMTS10 and fibrillin-1. Specifically, fibrillin-1 was investigated as a potential ADAMTS10 binding partner and substrate, and the role of ADAMTS10 in influencing microfibril biogenesis was addressed. Using ligand affinity blotting and surface plasmon resonance, recombinant ADAMTS10 was found to bind to fibrillin-1 with a high degree of specificity and with high affinity. Two sites of ADAMTS10 binding to fibrillin-1 were identified, one toward the N terminus and another in the C-terminal half of fibrillin-1. Confocal microscopy and immunoelectron microscopy localized ADAMTS10 to fibrillin-1-containing microfibrils in human tissues. Furin-activated ADAMTS10 could cleave fibrillin-1, but innate resistance of ADAMTS10 zymogen to propeptide excision by furin was observed, suggesting that, unless activated, ADAMTS10 is an inefficient fibrillinase. To investigate the role of ADAMTS10 in microfibril biogenesis, fetal bovine nuchal ligament cells were cultured in the presence or absence of ADAMTS10. Exogenously added ADAMTS10 led to accelerated fibrillin-1 microfibril biogenesis. Conversely, fibroblasts obtained from a Weill-Marchesani syndrome patient with ADAMTS10 mutations deposited fibrillin-1 microfibrils sparsely compared with unaffected control cells. Taken together, these findings suggest that ADAMTS10 participates in microfibril biogenesis rather than in fibrillin-1 turnover.

Monoclonal antibodies with defined recognition sequences in the stem region of CD44: detection of differential glycosylation of CD44 between tumor and stromal cells in tissue.

CD44 is an enigmatic cell adhesion molecule acting as a major receptor for hyaluronan and playing roles in many biological and pathological processes such as lymphocyte homing, T-cell activation, wound healing, angiogenesis, and metastatic spread of tumor cells. However, the complexity of the molecule, with its alternatively spliced variants, extensive glycosylation, and processing by different proteases, has hampered detailed analysis. In this study, we prepared four monoclonal antibodies (285-2F12, 284-43F1, 268-1F5, and 294-6F2) and one polyclonal antibody (C6) that recognize defined sequences in the stem region of CD44H. Interestingly, two of the monoclonal antibodies, 268-1F5 and 294-6F2, failed to recognize the CD44 expressed in five of the seven human tumor cell lines examined by Western blotting. Treatment of the samples with a combination of neuraminidase and O-glycosidase as well as the expression of mutants with site-directed mutations at possible modification sites rendered the CD44 reactive to the antibodies. Thus, the reactivity of the antibodies is sensitive to O-glycosylation presumably near the recognition sites. Glycosylation of CD44 that affects reactivity to the antibodies was found to be regulated differentially between tumor and stromal cells in two breast and three oral carcinoma tissues. Antibody 268-1F5 reacted to the tumor cells, but not to the cells in the surrounding stroma. On the other hand, the reactivity of 294-6F2 to the cells was opposite between the two tumor types. Thus, these sets of antibodies are useful to detect and analyze the as-yet-unknown roles of site-specific glycosylation of CD44, particularly in tumors.

Effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on migration of epidermal keratinocytes in vitro and wound healing in vivo.

Tissue inhibitors of metalloproteinases (TIMP), common inhibitors of matrix proteinases, have cell-promoting activity. We studied the effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on the migration of normal human epidermal keratinocytes (NHEK). An in vitro migration assay revealed that rh-TIMP-2 enhanced random migration (up to 170%, p<0.05) in a dose-dependent manner. When we applied rh-TIMP-2 solution (20 microg/20 microl/wound) daily to full-thickness wounds made with an 8-mm punch on the backs of healthy (n=8), aged (n=9), and diabetic (n=15) rodents, we observed faster wound closure (p<0.05) than in vehicle-treated controls. Accelerated wound closure was dose-dependent (0-20 microg/wound) in diabetic mice (n=6), and the optimal concentration was 10-20 microg of rh-TIMP-2/wound. Histological examinations performed on days 0, 5, 10, 15, and 20 in diabetic mice revealed faster migration of epidermal keratinocytes from wound edges. These results suggest that rh-TIMP-2 plays an important role in wound healing.

Inhibitory activity on matrix metalloproteinases and cell-proliferating activity of tissue inhibitor of metalloproteinases-1 (TIMP-1)-contrastive difference between human and bovine TIMP-1s on mouse cell proliferation.

Either human or bovine TIMP-1 inhibited matrix metalloproteinase (MMP) activities, such as collagenolytic, gelatinolytic and caseinolytic, expressed by mouse cells as well as those expressed by human cells. Bovine TIMP-1 stimulated the proliferation of both human and mouse cells, but human TIMP-1 only proliferate human cells and not mouse cells indicating that the cell-proliferating activity has more strict species specificity than MMP inhibitory activity. All the results from [(3)H]thymidine incorporation, the phosphorylation of tyrosine-containing cellular proteins and extracellular-signal-regulated protein kinases supported the cell-proliferating properties of both human and bovine TIMP-1s. Human TIMP-1 seems not to bind to TIMP-1 receptors on mouse cells.

Both tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 activate Ras but through different pathways.

Tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 have growth-stimulating activity for a wide range of cell types. Ras, which comprises a family of three members, i.e, Ha-Ras, Ki-Ras, and H-Ras, is known to participate in growth control in all its facets, including cell proliferation, transformation, differentiation, and apoptosis. In this study, we tested the hypothesis that Ras might be involved in the cell growth-promoting activity of TIMPs. Using MG-63 human osteosarcoma cells, we demonstrated that both TIMP-1 and TIMP-2 caused an increase in the Ras-GTP level in a dose-dependent manner. Our previous results indicated that TIMP-1 activity is mediated through the tyrosine kinase (TYK)/mitogen-activated protein kinase (MAPK) pathway. Here, we demonstrated that Ras activation by TIMP-1 was inhibited by a specific TYK inhibitor, herbimycin A, suggesting that the TYK/MAPK signaling pathway was involved in Ras activation by TIMP-1. However, the activation of Ras by TIMP-2 was inhibited by an inhibitor specific for cyclic AMP-dependent protein kinase (PKA), H89, suggesting the involvement of the PKA-mediated pathway. Furthermore, TIMP-2 promoted the formation of a complex between Ras-GTP and phosphoinositide 3-kinase.

Two-step sandwich enzyme immunoassay using monoclonal antibodies for detection of soluble and membrane-associated human membrane type 1-matrix metalloproteinase.

A two-step sandwich enzyme immunoassay (EIA) system for the detection of human membrane Type 1-matrix metalloproteinase (MT1-MMP) was established by using two monoclonal antibodies against recombinant MT1-MMP. MT1-MMP in which samples were reacted with solid-phase antibody and then detected with peroxidase-labeled second antibody. At least 1.25 ng/mL was detected by the EIA system, and linearity was obtained between 1.25 and 160 ng/mL. This EIA system is specific for MT1-MMP and did not show cross-reactivity against several other MMP's examined. Shedding of soluble MT1-MMP into the medium by some cancer cell lines was also detected by this system. However, soluble MT1-MMP in serum from normal and cancer patients was under the detection limit. Membrane-associated MT1-MMP of cancer cell lines was also detected after solubilization of the membranes with extraction buffer containing detergent. Additionally, MT1-MMP in clinical samples was examined. Elevated levels of MT1-MMP were detected in homogenate of cancer tissue compared with the levels for normal tissue and the level of MT1-MMP in tumors correlated with the rate of metastasis to the regional lymph nodes. Thus, we demonstrated that this EIA system is the first to measure MTI-MMP in clinical specimens, thus suggesting its useful for diagnosis of cancer or prediction of malignancy.