Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

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-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.     

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.     

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.     

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.     

Design, synthesis, and characterization of potent, slow-binding inhibitors that are selective for gelatinases.

Gelatinases have been shown to play a key role in angiogenesis and tumor metastasis. Small molecular weight synthetic inhibitors for these enzymes are highly sought for potential use as anti-metastatic agents. Virtually all of the known inhibitors of matrix metalloproteinases (MMPs) are broad spectrum. We report herein the synthesis and kinetic characterization of two compounds, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (compound 1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (compound 2), that are potent and selective gelatinase inhibitors. These compounds are slow, tight-binding inhibitors of gelatinases (MMP-2 and MMP-9) with K(i) values in the nanomolar range. In contrast, competitive inhibition of the catalytic domain of membrane-type 1 metalloproteinase (MMP-14(cat)) with comparable K(i) values (K(i) approximately 200 nm) was observed. Binding to stromelysin (MMP-3) was substantially weaker, with K(i) values in the micromolar range (K(i) approximately 10 microm). No binding to matrilysin (MMP-7) and collagenase 1 (MMP-1) was detected at inhibitor concentrations up to 60 microm. We have previously shown that synthetic MMP inhibitors work synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP in a process that depends on the affinity of the inhibitor toward MT1-MMP. It is shown herein that the dithiols are significantly less efficient (>100-fold) than marimastat, a broad-spectrum MMP inhibitor, in enhancing pro-MMP-2 activation in cells infected to express MT1-MMP, consistent with the lower affinity of the dithiols toward MT1-MMP. Thus, in contrast to broad-spectrum MMP inhibitors, the dithiols are less likely to promote MT1-MMP-dependent pro-MMP-2 activation in the presence of TIMP-2, while maintaining their ability to inhibit active MMP-2 effectively.     

Calcium regulation of matrix metalloproteinase-mediated migration in oral squamous cell carcinoma cells

Activation of matrix metalloproteinase 2 (MMP-2) has been shown to play a significant role in the behavior of cancer cells, affecting both migration and invasion. The activation process requires multimolecular complex formation involving pro-MMP-2, membrane type 1-MMP (MT1-MMP), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because calcium is an important regulator of keratinocyte function, we evaluated the effect of calcium on MMP regulation in an oral squamous cell carcinoma line (SCC25). Increasing extracellular calcium (0.09-1.2 mm) resulted in a dose-dependent increase in MT1-MMP-dependent pro-MMP-2 activation. Despite the requirement for MT1-MMP in the activation process, no changes in MT1-MMP expression, cell surface localization, or endocytosis were apparent. However, increased generation of the catalytically inactive 43-kDa MT1-MMP autolysis product and decline in the TIMP-2 levels in conditioned media were observed. The decrease in TIMP-2 levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that calcium promotes recruitment of TIMP-2 to MT1-MMP on the cell surface. Despite the decline in soluble TIMP-2, no accumulation of TIMP-2 in cell lysates was seen. Blocking TIMP-2 degradation with bafilomycin A1 significantly increased cell-associated TIMP-2 levels in the presence of high calcium. These data suggest that the decline in TIMP-2 is because of increased calcium-mediated MT1-MMP-dependent degradation of TIMP-2. In functional studies, increasing calcium enhanced MMP-dependent cellular migration on laminin-5-rich matrix using an in vitro colony dispersion assay. Taken together, these results suggest that changes in extracellular calcium can regulate post-translational MMP dynamics and thus affect the cellular behavior of oral squamous cell carcinoma.     

Membrane type 1-matrix metalloproteinase (MMP)-associated MMP-2 activation increases in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin

Gonadotropins stimulate ovarian proteolytic enzyme activity that is believed to be important for the remodeling of the follicular extracellular matrix. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified in vitro as an activator of pro-MMP-2 by forming a complex with tissue inhibitors of metalloproteinase-2 (TIMP-2). In the present study, the expression pattern of MT1-MMP mRNA and the role of MT1-MMP were examined in the ovary using the gonadotropin-treated immature rat model. Ovaries were collected at selected times after eCG or hCG. RNase protection assays revealed a transient increase in MT1-MMP mRNA beginning 4 h after hCG. High expression of MT1-MMP mRNA was localized to the theca-interstitial layer of developing and preovulatory follicles, while low expression was observed in the granulosa cell layer of developing follicles by in situ hybridization. The localization pattern of MT1-MMP mRNA was compared with TIMP-2 mRNA. Both MMP-2 and TIMP-2 mRNA were expressed in the theca layer of preovulatory follicles, showing a similarity to MT1-MMP mRNA expression. To further determine whether MT1-MMP activates pro-MMP-2 in the ovary, crude plasma membrane fractions from preovulatory ovaries were analyzed by gelatin zymography. In plasma membrane fractions, pro-MMP-2 increased around the time of ovulation. Upon incubation, pro-MMP-2 was activated with the highest levels of activation at 12 h post-hCG. The addition of MT1-MMP antibody or excess TIMP-2 to membrane fractions inhibited pro-MMP-2 activation. The increase in MT1-MMP mRNA may be an important part of the mechanism necessary for the efficient generation of active MMP-2 during the ovulatory process.     

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.     

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.     

Integrin cleavage facilitates cell surface-associated proteolysis required for vascular smooth muscle cell invasion

Vascular smooth muscle cell (VSMC) invasion is a key element in atherogenesis and restenosis, requiring integrins for adhesion/de-adhesion as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Among the MMP family, pro-MMP-2 is unique in its activation, depending on the formation of a multiprotein complex with MT1-MMP/TIMP-2 at the cell surface, in which integrin αvβ3 participates. Integrin αv and MT1-MMP are synthesized from precursors via furin-dependent cleavage of their pro-peptide. Furin is the prototypical proprotein convertase highly expressed in VSMCs and human atherosclerotic lesions. Its precise role in the tight network involving MMPs/integrins and their coordination and cooperation required for VSMC invasion is unknown. We demonstrate that furin-inhibition with decanoyl-RVKR-chloromethylketone inhibits VSMC invasion in a comparable degree to MMP inhibitors, which reduce the MT1-MMP–MMP-2 proteolytic cascade. Furin-inhibition did not prevent MT1-MMP/MMP-2 maturation. In contrast, it strongly reduced pro-αv cleavage, but did not lessen its cell membrane expression. However, inhibition of pro-αv processing via furin-inhibition strongly reduced pro-MMP-2 binding to the cell surface, thereby lessening its full maturation and diminishing the cell surface in situ proteolysis required for invasion. Thus, our data demonstrate a novel mechanism of furin-dependent αv cleavage that enhances pro-MMP-2 binding and activation at the cell membrane in cooperation with MT1-MMP in primary VSMCs. Processing of αv by furin contributes to the recruitment of enzymatic energy to the cell surface, thereby providing focalized proteolysis associated with VSMC invasion.     

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.     

Tissue inhibitor of metalloproteinases-2 (TIMP-2) suppresses TKR-growth factor signaling independent of metalloproteinase inhibition

The tissue inhibitors of metalloproteinases (TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell proliferation, migration, and invasion that are associated with extracellular matrix (ECM) turnover. Here we demonstrate a direct role for TIMP-2 in regulating tyrosine kinase-type growth factor receptor activation. We show that TIMP-2 suppresses the mitogenic response to tyrosine kinase-type receptor growth factors in a fashion that is independent of MMP inhibition. The TIMP-2 suppression of mitogenesis is reversed by the adenylate cyclase inhibitor SQ22536, and implicates cAMP as the second messenger in these effects. TIMP-2 neither altered the release of transforming growth factor alpha from the cell surface, nor epidermal growth factor (EGF) binding to the cognate receptor, EGFR. TIMP-2 binds to the surface of A549 cells in a specific and saturable fashion (K(d) = 147 pm), that is not competed by the synthetic MMP inhibitor BB-94 and is independent of MT-1-MMP. TIMP-2 induces a decrease in phosphorylation of EGFR and a concomitant reduction in Grb-2 association. TIMP-2 prevents SH2-protein-tyrosine phosphatase-1 (SHP-1) dissociation from immunoprecipitable EGFR complex and a selective increase in total SHP-1 activity. These studies represent a new functional paradigm for TIMP-2 in which TIMP suppresses EGF-mediated mitogenic signaling by short-circuiting EGFR activation.     

Type I collagen-induced MMP-2 activation coincides with up-regulation of membrane type 1-matrix metalloproteinase and TIMP-2 in cardiac fibroblasts

Migration of cardiac fibroblasts is implicated in infarct healing and ventricular remodeling. Activation of matrix metalloproteinases induced by three-dimensional type I collagen, the principal component of the myocardial interstitium, is hypothesized to be essential for this migration. By utilizing primary cultures of cardiac fibroblasts and collagen lattice models, we demonstrated that type I collagen induced MMP-2 activation, and cells undergoing a change from isometric tension to mechanical unloading were associated with increased levels of total and active MMP-2 species. The collagen-induced MMP-2 activation coincided with up-regulated cellular levels of both membrane type 1-matrix metalloproteinase (MT1-MMP) and TIMP-2. A fraction of cellular membrane prepared from cells embedded in the collagen lattice containing active MT1-MMP and TIMP-2 was capable of activating pro-MMP-2, and exogenous TIMP-2 had a biphasic effect on this membrane-mediated MMP-2 activation. Interestingly, the presence of 43-kDa MT1-MMP species in a fraction of intracellular soluble proteins prepared from monolayer cells but not cells embedded in the lattices indicates that MT1-MMP metabolizes differently under the two different culture conditions. Treatment of cells embedded in the lattice with furin inhibitor attenuated pro-MT1-MMP processing and MMP-2 activation and impeded cell migration and invasion. These results suggest that the migration and invasion of cardiac fibroblasts is furin-dependent and that the active species of MT1-MMP and MMP-2 may be involved in both events.     

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.     

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.     

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.