The complex between a tissue inhibitor of metalloproteinases (TIMP‐2) and 72‐kDa progelatinase is a metalloproteinase inhibitor

Abstract

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase.Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase.The progelatinase -TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.The tissue inhibitor of metalloproteinases (TIMP-1) is a glycoprotein with a molecular mass of about 30 kDa, purified initially from culture medium conditioned by normal skin fibroblasts, and later from various human sources, including plasma, amniotic fluid and synovial fluid [l -71.The TIMP-1 gene has been localized to the x chromosome [8]. The secreted protein consists of 184 amino acids and possesses six disulfide bonds and two glycosylation sites containing N-linked oligosaccharides [5, 91.TIMP-1 specifically inhibits the extracellular-matrix-degrading metalloproteinases such as collagenase, gelatinase and stromelysin. It has no activity on mammalian membrane metalloproteinase, procollagen peptidase, or on bacterial metalloproteinases such as thermolysin [lo].Originally it was thought that TIMP-1 only binds to active enzymes and not to latent ones. However, it was reported recently, that the 72-kDa progelatinase secreted by several cell types, such as Simian-virus-40-transformed human lung fibroblasts, Harvey murine sarcoma virus-transformed human bronchial epithelial cells, human A2058 melanoma cells and normal skin fibroblasts, exists in a stable, but noncovalent 1 : 1 stoichiometric complex with a 24-kDa inhibitor of metalloproteinases called TIMP-2 [ l l , 121. In addition, a 92-kDa progelatinase -TIMP-1 complex is secreted by human lung fibroblasts [13]. This proteinase is normally produced by macrophages and polymorphonuclear leukocytes (PMNL). PMNL do not produce a tissue inhibitor of metalloproteinases, which may account for its 10-fold-higher specific activity 1131.We found human rheumatoid synovial cells to secrete not only the 72-kDa-progelatinase -TIMP-2 complex and TIMP-1, but in addition free 72-kDa progelatinase. In this communication, we show that TIMP-2, although complexed with the progelatinase, still remains an inhibitor of metalloproteinases. MATERIAL AND METHODS Cell cultureHuman rheumatoid synovium obtained by surgery was washed in Hank's solution and freed from fat and cartilage, cut into pieces (approximately 1 mm3 in size), washed with Hank's solution again, and distributed into 75-cmZ culture flasks. 8 ml Dulbecco's modified Eagle's medium containing ...

A Cationic [60] Fullerene Derivative Reduces Invasion and Migration of HT-29 CRC Cells in Vitro at Dose Free of Significant Effects on Cell Survival

“…Moreover the complex pro-MMP2 and TIMP-2 is able to inhibit other MMPs such as MMP9 through the formation of a ternary complex pro-MMP2-TIMP-2-MMP9 [23][24][25]. This data suggest that the reduced MMP9 activity detected in treated HT-29 cells, could be due to the increased level of TIMP-2 after C 60 + treatment and its modulation on MMP9 activity via MMP2 involvement.…”

Characterization of a Gelatinase from Human Rheumatoid Synovial Fluid Cells

“…Both enzymes (leukocyte and fibroblast gelatinase) also show different behaviour against the tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). While TIMP-1 exclusively binds to the latent leukocyte gelatinase, TIMP-2 exclusively binds to the latent fibroblast gelatinase in a l : l stoichiometry (8,(34)(35)(36). The activated enzymes are inhibited by both TIMPs.…”

Substance P Induces the Secretion of Gelatinase A from Human Synovial Fibroblasts

“…Gelatinase A and stromelysin-1 were isolated in our laboratory as previously described (17,18). Gelatinase B was purified from buffy coat essentially as described (19).…”

“…It is known that human synovial fibroblasts secrete progelatinase A mainly complexed with the tissue inhibitor 2 ) of metalloproteinases TIMP-2. After activation the whole complex displays gelatinolytic activity, while progelatinase A-TIMP-2 is an inhibitor of other matrix metalloproteinases as well (17). TIMP-2 is visible in gel electrophoresis only after silver staining (not shown).…”