Characterization of a rainbow trout matrix metalloproteinase capable of degrading type I collagen.

Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP-13 is well-known as collagenase-3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP-13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino-acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP-13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.     

Characterization of the mechanisms by which gelatinase A, neutrophil collagenase, and membrane-type metalloproteinase MMP-14 recognize collagen I and enzymatically process the two alpha-chains

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.     

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.     

Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.     

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.     

Method to analyze collagenase and gelatinase activity by fibroblasts in culture

Summary The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)NH₂, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.     

Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3

During tissue-invasive events, migrating cells penetrate type I collagen-rich interstitial tissues by mobilizing undefined proteolytic enzymes. To screen for members of the matrix metalloproteinase (MMP) family that mediate collagen-invasive activity, an in vitro model system was developed wherein MDCK cells were stably transfected to overexpress each of ten different MMPs that have been linked to matrix remodeling states. MDCK cells were then stimulated with scatter factor/hepatocyte growth factor (SF/HGF) to initiate invasion and tubulogenesis atop either type I collagen or interstitial stroma to determine the ability of MMPs to accelerate, modify, or disrupt morphogenic responses. Neither secreted collagenases (MMP-1 and MMP-13), gelatinases (gelatinase A or B), stromelysins (MMP-3 and MMP-11), or matrilysin (MMP-7) affected SF/HGF-induced responses. By contrast, the membrane-anchored metalloproteinases, membrane-type 1 MMP, membrane-type 2 MMP, and membrane-type 3 MMP (MT1-, MT2-, and MT3-MMP) each modified the morphogenic program. Of the three MT-MMPs tested, only MT1-MMP and MT2-MMP were able to directly confer invasion-incompetent cells with the ability to penetrate type I collagen matrices. MT-MMP-dependent invasion proceeded independently of proMMP-2 activation, but required the enzymes to be membrane-anchored to the cell surface. These findings demonstrate that MT-MMP-expressing cells can penetrate and remodel type I collagen-rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases.     

MMP-1: the elder of the family

The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue remodeling. Human fibroblast collagenase (MMP-1) was the first vertebrate collagenase purified as a protein and cloned as a cDNA, and is considered the prototype for all the interstitial collagenases. It is synthesized as a zymogen where N-terminal residues are removed by proteolysis and shares with other MMPs a catalytic domain and a carboxy terminal domain with sequence similarity to hemopexin. Importantly, MMP-1 should be considered a multifunctional molecule since it participates not only in the turnover of collagen fibrils in the extracellular space but also in the cleavage of a number of non-matrix substrates and cell surface molecules suggesting a role in the regulation of cellular behaviour. Furthermore, an extensive body of evidence indicates that MMP-1 plays an important role in diverse physiologic processes such as development, tissue morphogenesis, and wound repair. Likewise, it seems to be implicated in a variety of human diseases including cancer, rheumatoid arthritis, pulmonary emphysema and fibrotic disorders, suggesting that its inhibition or stimulation may open therapeutic avenues.     

Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.     

Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone

There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.     

Dynamics of matrix metalloproteinases activity of primary murine embryonic fibroblasts during cultivation

Embryonic cells regulate the expression of matrix metalloproteinases (MMP) providing remodulation of extracellular matrix, which in turn provides the changes in cell adhesion and migration during the cell development and differentiation. In present work we studied the changes of gelatinases (MMP-2 and MMP-9) and collagenases (MMP-1 and MMP-8) activities in the process of cultivating the primary murine embryonic fibroblasts (MEF). Cultivation was continued for 6 passages, after that the culture died in time. According to gelatin and collagen zymography results, drastic changes of all MMPs activities occurred during the third passage of cell cultivation. The MMP-1 and MMP-9 activity appears in the middle of cultivation and then disappeared at the end. The most important event MEF cultivation is appearance and subsequent reservation of collagenase MMP-8 and active form of gelatinase MMP-2.     

Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3)

We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.     

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.     

Human tissue factor pathway inhibitor-2 does not bind or inhibit activated matrix metalloproteinase-1

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor associated with the extracellular matrices of vascular cells. A recent report provided in vitro evidence that TFPI-2 may be a novel inhibitor of the matrix metalloproteinases MMP-1, MMP-13, MMP-2 and MMP-9. In studies aimed at identifying the structural elements of TFPI-2 mediating the putative inhibition of the above MMPs, we re-examined the ability of native TFPI-2 to form complexes with MMP-2, MMP-9 and MMP-1, as well as assess its ability to inhibit the proteolytic activity of the interstitial collagenase, activated MMP-1. We report here that TFPI-2 failed to form complexes with MMP-2, MMP-9 and MMP-1 as revealed in immunoprecipitation and ligand blotting studies. In addition, TFPI-2 had no influence on the proteolytic activity of activated MMP-1 towards triple-helical collagen. These data provide presumptive evidence that TFPI-2 does not bind to MMP-2, MMP-9 and MMP-1, or regulate MMP-1, in the extracellular matrix.     

The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.     

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.     

Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis

Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. In the acute phases of liver injury and as liver fibrosis progresses, there is increased expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Among the changes described, striking features include increased expression of gelatinase A (MMP-2) and membrane type 1-MMP (MT(1)-MMP; MMP-14) as well as TIMP-1 and TIMP-2. These molecules and other family members are involved in regulating degradation of both normal and fibrotic liver matrix. This article outlines recent progress in this field and discusses the mechanisms by which MMPs and TIMPs may contribute to the progression and regression of liver fibrosis. Recently described properties of MMPs and TIMPs of relevance to the pathogenesis of liver fibrosis are outlined. The proposal that regression of liver fibrosis is mediated by decreased expression of TIMPs and involves degradation of fibrillar collagens by a combination of MT(1)-MMP and gelatinase A, in addition to interstitial collagenase, is explored.