Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

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.     

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.     

Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis

Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.     

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.     

Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion

Activation of proMMP-2 by MT1-MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP-2 bound to MT1-MMP on the cell surface acts as a receptor for proMMP-2. Subsequently, adjacent TIMP-2-free MT1-MMP activates the proMMP-2 in the ternary complex. In this study, we demonstrate that MT1-MMP forms a homophilic complex through the hemopexin-like (PEX) domain that acts as a mechanism to keep MT1-MMP molecules close together to facilitate proMMP-2 activation. Deletion of the PEX domain in MT1-MMP, or swapping the domain with the one derived from MT4-MMP, abolished the ability to activate proMMP-2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1-MMP lacking the catalytic domain (MT1PEX-F) efficiently inhibited complex formation of the full-length enzymes and activation of pro MMP-2. Furthermore, expression of MT1PEX-F inhibited proMMP-2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1-MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue.     

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.     

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.     

Flexibility and variability of TIMP binding: X-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.     

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.     

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.     

Contrasting expression of membrane metalloproteinases, MT1-MMP and MT3-MMP, suggests distinct functions in skeletal development

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is the most ubiquitous and widely studied of the membrane-type metalloproteinases (MT-MMPs). It was thus surprising to find no published data on chicken MT1-MMP. We report here the characterization of the chicken gene. Its low sequence identity with the MT1-MMP genes of other species, high GC content, and divergent catalytic domain explains the absence of data and our difficulties in characterizing the gene. The absence of structural features in the chicken gene that have been suggested to be critical for the activation of MMP-2 by MT1-MMP; for the effect of MT1-MMP on cell migration and for the recycling of MT1-MMP suggest these features are either not essential or that MT1-MMP does not perform these functions in chickens. Comparison of the expression of chicken MT1-MMP with MT3-MMP and with MMP-2 and MMP-13 has confirmed the previously recognized co-expression of MT1-MMP with MMP-2 and MMP-13 in fibrous and vascular tissues, particularly those surrounding the developing long bones in other species. By contrast, MT3-MMP expression differs markedly from that of MT1-MMP and of both MMP-2 and MMP-13. MT3-MMP is expressed by chondrocytes of the developing articular surface. Similar expression patterns of this group of MT-MMPs and MMPs have been observed in mouse embryos and suggest distinct and specific functions for MT1-MMP and MT3-MMP in skeletal development.     

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.     

Expanding the Activity of Tissue Inhibitors of Metalloproteinase (TIMP)-1 against Surface-Anchored Metalloproteinases by the Replacement of Its C-Terminal Domain: Implications for Anti-Cancer Effects

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, we replaced the C-terminal domain of TIMP-1 with those of TIMP-2, -3 and -4 to create a series of “T1:TX” chimeras. The affinity of the chimeras against ADAM10, ADAM17, MMP14 and MMP19 was investigated. We can show that replacement of the C-terminal domain by those of other TIMPs dramatically increased the affinity of TIMP-1 for some MPs. Furthermore, the chimeras were able to suppress TNF-α and HB-EGF shedding in cell-based setting. Unlike TIMP-1, T1:TX chimeras had no growth-promoting activity. Instead, the chimeras were able to inhibit cell migration and development in several cancer cell lines. Our findings have broadened the prospect of TIMPs as cancer therapeutics. The approach could form the basis of a new strategy for future TIMP engineering.     

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.     

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.