Mechanism Of Cell Surface Activation Of 72-kDa Type IV Collagenase

Abstract

Matrix metalloproteases are secreted by mammalian cells as zymogens and, upon activation, initiate tissue remodeling by proteolytic degradation of collagens and proteoglycans. Activation of the secreted proenzymes and interaction with their specific inhibitors determine the net enzymatic activity in the extracellular space. We have previously demonstrated that 72T4Cl can be activated by a plasma membrane-dependent mechanism specific for this enzyme. Here, we report purification of the membrane activator of 72T4Cl, which is a new metalloprotease identical to a recently cloned membrane-type matrix metalloprotease (MT-MMP). We demonstrate that activated MT-MMP acts as a cell surface tissue inhibitor of metalloprotease 2 (TIMP-2) receptor with Kd = 2.54 x 10(-9) M. The activator.TIMP-2 complex in turn acts as a receptor for 72T4Cl (Kd = 0.56 x 10(-9) M, binding to the carboxyl-end domain of the enzyme. Activation of 72T4Cl on the cell membrane provides a basic mechanism for spatially regulated extracellular proteolysis and presents a new target for prognosis and treatment of metastatic disease. The activation, purified as a tri-molecular complex of MT-MMP.TIMP2.carboxyl-end domain of 72T4Cl, is itself an activated form of MT-MMP, posing the following question: what is the mechanism of the activator's activation?

Furin regulates the intracellular activation and the uptake rate of cell surface-associated MT1-MMP

“…According to the N-terminal sequencing of cellular MT1-MMP, the Tyr 112 -species represents the fully mature enzyme (Strongin et al, 1995). The Cys 93 -MT1-MMP cellular intermediate remains undetected as of this report.…”

“…Furin cleavages lead to the activation and to the presentation of the functionally active, commencing at Tyr 112 , mature MT1-MMP at the cell surfaces (Strongin et al, 1995;Osenkowski et al, 2004). There is evidence that a portion of the cellular MT1-MMP pool is processed by additional PCs and also by autocatalysis directly at the cell surface (Sato et al, 1999;Yana and Weiss, 2000;Rozanov and Strongin, 2003;Deryugina et al, 2004).…”

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

“…using a carboxyl terminal fragment of gelatinase A and suggested that MT1-MMP-TIMP-2 complex is the receptor and activator of gelatinase A [18]. However, we observed that MT1-MMP expressed in COS-1 cells was substantially free of TIMP-2 as assessed by immunoprecipitation using a monoclonal antibody against TIMP-2 and exogenously added TIMP-2 bound to these cells which abolished binding of progelatinase A (data not shown).…”

“…Progelatinase A is unique among matrix metalloproteinases, in that it is not activated after digestion with exogenous proteinases such as plasmin, plasma kallikrein, neutrophil elastase or cathepsin G, which are putative physiological activators of other members of this family [4]. Progelatinase A is specifically activated on the surface of tumor and normal fibroblast cells exposed to 12-O-tetradecanoylphorbol acetate (TPA) or concanavalin A, as a result of proteolysis, and it is mediated by a fraction of the cell membrane that is sensitive to metalloproteinase inhibitors, including tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) [5][6][7][8]. It has also been demonstrated that the activated form of gelatinase A is enriched in the plasma membranes of cancer cells particularly at the invadopodia which is the site of cellular invasion [9][10][11][12][13].…”

Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins

“…[22][23][24][25] Higher concentrations of TIMP-2, but not TIMP-1, inhibit MT1-MMP. 26,27 In a recent study of adenovirus-mediated TIMP gene transfer into isolated SMC, 19 TIMP-2, but not TIMP-1, was shown to inhibit proliferation, an effect not mediated by inhibition of MMPs. On the other hand, growth promoting effects of TIMP-2 on HT1080 fibrosarcoma, normal dermal fibroblasts and Raji lymphoma cells have been described.…”