Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation

Previous studies have shown that membrane type 1-matrix metalloproteinase (MT1-MMP) (MMP-14) initiates pro-MMP-2 activation in a process that is tightly regulated by the level of tissue inhibitor of metalloproteinase (TIMP)-2. However, given the difficulty in modulating TIMP-2 levels, the direct effect of TIMP-2 on MT1-MMP processing and on pro-MMP-2 activation in a cellular system could not be established. Here, recombinant vaccinia viruses encoding full-length MT1-MMP or TIMP-2 were used to express MT1-MMP alone or in combination with various levels of TIMP-2 in mammalian cells. We show that TIMP-2 regulates the amount of active MT1-MMP (57 kDa) on the cell surface whereas in the absence of TIMP-2 MT1-MMP undergoes autocatalysis to a 44-kDa form, which displays a N terminus starting at Gly(285) and hence lacks the entire catalytic domain. Neither pro-MT1-MMP (N terminus Ser(24)) nor the 44-kDa form bound TIMP-2. In contrast, active MT1-MMP (N terminus Tyr(112)) formed a complex with TIMP-2 suggesting that regulation of MT1-MMP processing is mediated by a complex of TIMP-2 with the active enzyme. Consistently, TIMP-2 enhanced the activation of pro-MMP-2 by MT1-MMP. Thus, under controlled conditions, TIMP-2 may act as a positive regulator of MT1-MMP activity by promoting the availability of active MT1-MMP on the cell surface and consequently, may support pericellular proteolysis.     

Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (Membrane type 1)-MMP-dependent activation of pro-MMP-2

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.     

MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (K(i) = 0.2 nm) over TIMP-2 (K(i) = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.     

Distinct roles of catalytic and pexin-like domains in membrane-type matrix metalloproteinase (MMP)-mediated pro-MMP-2 activation and collagenolysis

Members of the membrane-type matrix metalloproteinase (MT-MMPs) family are dual regulators of extracellular matrix remodeling through direct degradation of extracellular matrix components and activation of other latent MMPs. However, the structural basis of this functional diversity remains poorly understood. In an attempt to dissect the structural determinants for MT-MMP function, we performed domain exchange experiments between MT1-MMP and its close relative MT3-MMP and analyzed the exchange chimeras for pro-MMP-2 activation and collagen degradation at the cellular level. Our results indicate that catalytic domains determine the pattern of pro-MMP-2 activation, whereas pexin-like domains modulate the level of activation. On the other hand, both the catalytic and pexin-like domains of MT1-MMP are required for strong collagenolysis because exchanging either domain with that of MT3-MMP yielded significantly lower activity, and the introduction of the MT1-MMP catalytic or pexin-like domain into MT3-MMP failed to generate any significant enhancement of collagenolytic activity compared with wild-type MT3-MMP. Interestingly, the cytoplasmic domain of MT1-MMP behaves as a negative regulator not only for MT1-MMP itself, but also for MT3-MMP in both pro-MMP-2 activation and collagenolysis, consistent with and extending our recent findings (Jiang, A., Lehti, K., Wang, X., Weiss, S. J., Keski-Oja, J., and Pei, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13693-13698). Taken together, these results demonstrate that domains in MT-MMPs function differently toward a given substrate and thus should be targeted differentially for future therapeutic development.     

Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes

MMP-9 (gelatinase B) is produced in a latent form (pro-MMP-9) that requires activation to achieve catalytic activity. Previously, we showed that MMP-2 (gelatinase A) is an activator of pro-MMP-9 in solution. However, in cultured cells pro-MMP-9 remains in a latent form even in the presence of MMP-2. Since pro-MMP-2 is activated on the cell surface by MT1-MMP in a process that requires TIMP-2, we investigated the role of the MT1-MMP/MMP-2 axis and TIMPs in mediating pro-MMP-9 activation. Full pro-MMP-9 activation was accomplished via a cascade of zymogen activation initiated by MT1-MMP and mediated by MMP-2 in a process that is tightly regulated by TIMPs. We show that TIMP-2 by regulating pro-MMP-2 activation can also act as a positive regulator of pro-MMP-9 activation. Also, activation of pro-MMP-9 by MMP-2 or MMP-3 was more efficient in the presence of purified plasma membrane fractions than activation in a soluble phase or in live cells, suggesting that concentration of pro-MMP-9 in the pericellular space may favor activation and catalytic competence.     

Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases

Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.     

TIMP independence of matrix metalloproteinase (MMP)-2 activation by membrane type 2 (MT2)-MMP is determined by contributions of both the MT2-MMP catalytic and hemopexin C domains

The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP.MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2.     

Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous modulators of the zinc-dependent mammalian matrix metalloproteinases (MMPs) and their close associates, proteinases of the ADAM (a disintegrin and metalloproteinase) and ADAM with thrombospondin repeats families. There are four variants of TIMPs, and each has its defined set of metalloproteinase (MP) targets. TIMP-1, in particular, is inactive against several of the membrane-type MMPs (MT-MMPs), MMP-19, and the ADAM proteinase TACE (tumor necrosis factor-alpha-converting enzyme, ADAM-17). The molecular basis for such inactivity is unknown. Previously, we showed that TIMP-1 could be transformed into an active inhibitor against MT1-MMP by the replacement of threonine 98 residue with leucine (T98L). Here, we reveal that the T98L mutation has in fact transformed TIMP-1 into a versatile inhibitor against an array of MPs otherwise insensitive to wild-type TIMP-1; examples include TACE, MMP-19, and MT5-MMP. Using T98L as the scaffold, we created a TIMP-1 variant that is fully active against TACE. The binding affinity of the mutant (V4S/TIMP-3-AB-loop/V69L/T98L) (K (app)(i) 0.14 nm) surpassed that of TIMP-3 (K (app)(i) 0.22 nm), the only natural TIMP inhibitor of the enzyme. The requirement for leucine is absolute for the transformation in inhibitory pattern. On the other hand, the mutation has minimal impact on the MPs already well inhibited by wild-type TIMP-1, such as gelatinase-A and stromelysin-1. Not only have we unlocked the molecular basis for the inactivity of TIMP-1 against several of the MPs, but also our findings fundamentally modify the current beliefs on the molecular mechanism of TIMP-MP recognition and selectivity.     

The inactive 44-kDa processed form of membrane type 1 matrix metalloproteinase (MT1-MMP) enhances proteolytic activity via regulation of endocytosis of active MT1-MMP

Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly(285)-Val(582)) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast, deletion of the hinge turns the 44-MT1 form into a negative regulator of enzyme function in vitro and in vivo, suggesting a key role for the hinge region in the functional relationship between active and processed MT1-MMP. Together, these results suggest a novel role for the 44-kDa form of MT1-MMP generated during autocatalytic processing in maintaining the pool of active enzyme at the cell surface.     

Differential roles of TIMP-4 and TIMP-2 in pro-MMP-2 activation by MT1-MMP

The tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of MMP enzymatic activity. However, TIMP-2 can promote the activation of pro-MMP-2 by MT1-MMP. This process is mediated by the formation of a complex between MT1-MMP, TIMP-2, and pro-MMP-2. Binding of TIMP-2 to active MT1-MMP also inhibits the autocatalytic turnover of MT1-MMP on the cell surface. Thus, under certain conditions, TIMP-2 is a positive regulator of MMP activity. TIMP-4, a close homologue of TIMP-2 also binds to pro-MMP-2 and can potentially participate in pro-MMP-2 activation. We coexpressed MT1-MMP with TIMP-4 and investigated its ability to support pro-MMP-2 activation. TIMP-4, unlike TIMP-2, does not promote pro-MMP-2 activation by MT1-MMP. However, TIMP-4 binds to MT1-MMP inhibiting its autocatalytic processing. When coexpressed with TIMP-2, TIMP-4 competitively reduced pro-MMP-2 activation by MT1-MMP. A balance between TIMP-2 and TIMP-4 may be a critical factor in determining the degradative potential of cells in normal and pathological conditions.     

Membrane type-1 matrix metalloproteinase and TIMP-2 in tumor angiogenesis.

The matrix metalloproteinases (MMPs) constitute a multigene family of over 23 secreted and cell-surface associated enzymes that cleave or degrade various pericellular substrates. In addition to virtually all extracellular matrix (ECM) compounds, their targets include other proteinases, chemotactic molecules, latent growth factors, growth factor-binding proteins and cell surface molecules. The MMP activity is controlled by the physiological tissue inhibitors of MMPs (TIMPs). There is much evidence that MMPs and their inhibitors play a key role during extracellular remodeling in physiological situations and in cancer progression. They have other functions that promoting tumor invasion. Indeed, they regulate early stages of tumor progression such as tumor growth and angiogenesis. Membrane type MMPs (MT-MMPs) constitute a new subset of cell surface-associated MMPs. The present review will focus on MT1-MMP which plays a major role at least, in the ECM remodeling, directly by degrading several of its components, and indirectly by activating pro-MMP2. As our knowledge on the field of MT1-MMP biology has grown, the unforeseen complexities of this enzyme and its interaction with its inhibitor TIMP-2 have emerged, often revealing unexpected mechanisms of action.     

Membrane type 1 matrix metalloproteinase-associated degradation of tissue inhibitor of metalloproteinase 2 in human tumor cell lines

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been shown to function as a receptor for secreted pro-MMP-2, resulting in the formation of a trimolecular complex. In the presence of uncomplexed active MT1-MMP, the prodomain of cell surface-associated MMP-2 is cleaved, and activated MMP-2 is released. However, the behavior of MT1-MMP-bound TIMP-2 during MMP-2 activation is currently unknown. In this study, (125)I-labeled recombinant TIMP-2 ((125)I-rTIMP-2) was used to investigate the fate of TIMP-2 during pro-MMP-2 activation by HT1080 and transfected A2058 cells. HT1080 and A2058 cells transfected with MT1-MMP cDNA (but not vector-transfected A2058 cells) were able to bind (125)I-rTIMP-2, to activate pro-MMP-2, and to process MT1-MMP into an inactive 43-kDa form. Under these conditions, (125)I-rTIMP-2 bound to the cell surface was rapidly internalized and degraded in intracellular organelles through a bafilomycin A(1)-sensitive mechanism, and (125)I-bearing low molecular mass fragment(s) were released in the culture medium. These different processes were inhibited by hydroxamic acid-based synthetic MMP inhibitors and rTIMP-2, but not by rTIMP-1 or cysteine, serine, or aspartic proteinase inhibitors. These results support the concept that the MT1-MMP-dependent internalization and degradation of TIMP-2 by some tumor cells might be involved in the regulation of pericellular proteolysis.     

Individual Timp deficiencies differentially impact pro-MMP-2 activation

Membrane-type matrix metalloproteinases (MT-MMPs) have emerged as key enzymes in tumor cell biology. The importance of MT1-MMP, in particular, is highlighted by its ability to activate pro-MMP-2 at the cell surface through the formation of a trimolecular complex comprised of MT1-MMP/tissue inhibitor of metalloproteinase-2 (TIMP-2)/pro-MMP-2. TIMPs 1-4 are physiological MMP inhibitors with distinct roles in the regulation of pro-MMP-2 processing. Here, we have shown that individual Timp deficiencies differentially affect MMP-2 processing using primary mouse embryonic fibroblasts (MEFs). Timp-3 deficiency accelerated pro-MMP-2 activation in response to both cytochalasin D and concanavalin A. Exogenous TIMP-2 and N-TIMP-3 inhibited this activation, whereas TIMP-3 containing matrix from wild-type MEFs did not rescue the enhanced MMP-2 activation in Timp-3(-/-) cells. Increased processing of MMP-2 did not arise from increased expression of MT1-MMP, MT2-MMP, or MT3-MMP or altered expression of TIMP-2 and MMP-2. To test whether increased MMP-2 processing in Timp-3(-/-) MEFs is dependent on TIMP-2, double deficient Timp-2(-/-)/-3(-/-) MEFs were used. In these double deficient cells, the cleavage of pro-MMP-2 to its intermediate form was substantially increased, but the subsequent cleavage of intermediate-MMP-2 to fully active form, although absent in Timp-2(-/-) MEFs, was detectable with combined Timp-2(-/-)/-3(-/-) deficiency. TIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.     

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located ''receptor'' involved in pro-MMP-2 activation. We have solved the 2.75 A crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the beta-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1'' specificity pocket of cdMT1-MMP, with its Ogamma pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions.     

Characterization of the AB loop region of TIMP-2. Involvement in pro-MMP-2 activation

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is unique as it is the only member of the TIMP family that is involved in the cellular activation of promatrix metalloproteinase-2 (pro-MMP-2) by virtue of forming a trimolecular complex with membrane type 1 matrix metalloproteinase (MT1-MMP) on the cell surface. TIMP-4 is similar in structure to TIMP-2 but is unable to support the activation of the proenzyme. Several reports have highlighted the importance of the TIMP-2 C-terminal domain in the pro-MMP-2 activation complex; however, very little is known about the role of the extended AB loop of TIMP-2 in this mechanism even though it has been shown to interact with MT1-MMP. In this study we show by mutagenesis and kinetic analysis that it is possible to transfer the MT1-MMP binding affinity of the TIMP-2 AB loop to TIMP-4 but that its transplantation into TIMP-4 does not endow the inhibitor with pro-MMP-2 activating activity. However, transfer of both the AB loop and C-terminal domain of TIMP-2 to TIMP-4 generates a mutant that can activate pro-MMP-2 and so demonstrates that both these regions of TIMP-2 are important for the activation process.     

Biochemical characterization of the catalytic domain of membrane-type 4 matrix metalloproteinase.

A C-terminal truncated form of membrane-type 4 matrix metalloproteinase (MT4-MMP; MMP 17), lacking the hemopexin-like and transmembrane domain, was expressed in Escherichia coli. The catalytic domain was produced by tryptic activation of the recombinant proenzyme and proved to be catalytically active towards the fluorogenic substrate for matrix metalloproteinases (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu(3-(2,4-dinitrophenyl)-L-2,3-diaminopro-p ionyl)-Ala-Arg-NH2. In contrast to the other three MT-MMPs (MT1-, MT2-, and MT3-MMP), the catalytic domain of MT4-MMP does not activate progelatinase A, nor does it hydrolyze one of the offered extracellular matrix (ECM) proteins, such as collagen types I, II, III, IV, and V, gelatin, fibronectin, laminin or decorin. TIMP-1, a poor inhibitor of MT1-, MT2- and MT3-MMP, suppresses MT4-MMP activity effectively. The progelatinase A/TIMP-2 complex that usually reacts like TIMP-2 also inhibits MT4-MMP. TIMP-2, a strong inhibitor of other MT-MMPS, inhibits MT4-MMP at low concentrations. With increasing TIMP-2 concentration, however, activity passes through a minimum and then increases until at high TIMP-2 concentration the activity is the same as in the absence of TIMP-2. TIMP-1 or the progelatinase A/TIMP-2 complex do not prevent reactivation of MT4-MMP catalytic domain at high TIMP-2 concentrations.     

Functional interplay between type I collagen and cell surface matrix metalloproteinase activity

Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves beta(1) integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both alpha(2)beta(1) and alpha(3)beta(1) integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of alpha(3)beta(1) strongly promotes activation of pro-MMP-2, whereas alpha(2)beta(1) integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance.     

Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.