Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonary artery smooth muscle. This inhibitor was purified by ammonium sulfate precipitation followed by gelatin sepharose and lentil lectin sepharose affinity chromatography and continuous elution electrophoresis by Prep Cell Model 491 (Bio-Rad, USA). SDS-PAGE revealed that the inhibitor has an apparent molecular mass of 21 kDa and was confirmed as TIMP-2 by (i) Western immunoblot assay using bovine polyclonal TIMP-2 antibody; and also by (ii) amino terminal amino acid sequence analysis of the purified inhibitor is found to be identical with TIMP-2 obtained from other sources. The purified 21 kDa inhibitor was found to be active against matrix metalloproteinase-2 (MMP-2, 72 kDa gelatinase) and matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase), the ambient MMPs in the pulmonary artery smooth muscle. The inhibitor was also found to be sensitive to the activated 72 kDa gelatinase-TIMP-2 complex and also active human interstitial collagenase. By contrast, it was found to be insensitive to the serine proteases: trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation. Treatment of the inhibitor with hydrogen peroxide, superoxide generating system (hypoxanthine plus xanthine oxidase) and peroxynitrite inactivated the inhibitor.     

Role of membrane-associated CA2+ dependent matrix metalloprotease-2 in the oxidant activation of Ca2+ATPase by tertiary butylhydroperoxide

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca²⁺⁺ATPase activity. The smooth muscle plasma membrane possesses a Ca²⁺⁺ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca²⁺⁺ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca²⁺⁺ dependent protease that degrades ¹⁴C-gelatin. The oxidant triggered protease activity and the Ca²⁺⁺ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca²⁺⁺ dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca²⁺⁺ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca²⁺⁺ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca²⁺⁺ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca²⁺⁺ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH.     

Native TIMP-free 70 kDa progelatinase (MMP-2) secreted at elevated levels by RSV transformed fibroblasts

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS-PAGE and nondenaturing, native gel electrophoresis. The purified TIMP-free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP-free progelatinase with the organomercurial, p-aminophenylmercuric acetate (APMA), results in rapid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP-complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the specific enzyme activity of the preparation is 8 to 18-fold lower. Purified avian TIMP-2 added to the TIMP-free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP-free progelatinase than the normal CEF cultures which produce predominantly TIMP-complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapid activation by TIMP-2, and the potential effect of the altered balance between TIMP-free and TIMP-complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed. © 1994 Wiley-Liss, Inc.     

Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2

To study the activation of human 72-kDa gelatinase, and its relation to tissue inhibitor of metalloproteinases 2 (TIMP-2), we purified human 72-kDa progelatinase both as a complex with TIMP-2 and as a free proteinase. Activation of progelatinase-TIMP-2 complexes with 4-aminophenylmercuric acetate yielded gelatinolytically active enzyme migrating at 62 kDa. TIMP-2 remained bound to the active enzyme. Removal of TIMP-2 from progelatinase by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid, followed by complete dialysis in neutral pH buffer, resulted in multiple fragments. These fragments were formed as a result of the cleavage of 72-kDa progelatinase at several locations. Cleavage at the amino terminus was restricted to the removal of the propeptide, except in the case of degradation leading to inactive fragments. Two active species autocatalytically evolved upon removal of TIMP-2 from progelatinase. The 62 kDa-activated gelatinase lacked the amino-terminal propeptide, which is known to be removed upon treatment with 4-aminophenylmercuric acetate. In addition, an active 42.5-kDa fragment lacking both the propeptide and a portion of the carboxyl terminus was formed. This low-molecular-weight active form of 72-kDa progelatinase retained its ability to bind and degrade gelatin. Self-activation and degradation of 72-kDa progelatinase can be prevented by agents that inhibit metalloproteinases, including 1,10-phenanthroline. Evidence presented here suggests that TIMP-2 binds to a stabilization site that is independent of the active site. This stabilization site does not bind TIMP-1 (TIMP). Occupation of this site by TIMP-2 prevents autocatalytic activation and degradation but does not prevent gelatinolysis by the enzyme-inhibitor complex.     

The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase. Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase. The progelatinase-TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.     

Matrix metalloprotease 2-mediated activation of Ca(2+)-ATPase by superoxide radical (O2*-) in plasma membrane of bovine pulmonary vascular smooth muscle

The role of the matrix metalloprotease-2 (MMP-2) in regulating Ca(2+)-ATPase activity in bovine pulmonary artery smooth muscle plasma membranes during treatment with the O2*- generating system, hypoxanthine (HPX) plus xanthine oxidase (XO) has been studied. The smooth muscle membranes possess matrix metalloprotease (MMP) activity in gelatin zymogram, having an apparent molecular mass of 72 kDa; the activity is inhibited by the tissue inhibitor of metalloprotease-2 (TIMP-2). Since both protease and MMP-2 have same molecular mass and are inhibited by TIMP-2, it may, therefore, be suggested that the protease is the MMP-2. Treatment of the smooth muscle membrane suspension with the O2*- generating system stimulates MMP-2 activity, as evidenced by an apparent increase in the intensity of the protease activity. O2*- also enhances [14C]-gelatin degradation and Ca(2+)-ATPase activity. The increase in MMP activity, assessed by [14C]-gelatin degradation and Ca(2+)-ATPase activity are inhibited upon pretreatment with superoxide dismutase (SOD). The O2*- triggered MMP and Ca(2+)-ATPase activities in the membrane are found to be inhibited by TIMP-2. The stimulation of the MMP and Ca(2+)-ATPase activities remain unaffected by the inhibitors of serine, thiol and cysteine groups of proteases such as phenylmethylsulfonylfluoride (PMSF), Bowman Birk inhibitor (BBI), chymostatin, N-ethylmaleimide, leupeptin, antipain and pepstatin. Adding pure bovine MMP-2 to the smooth muscle membrane suspension causes an increase in Ca(2+)-ATPase activity, but the pretreatment with TIMP-2 inhibits the increase in the enzyme activity.     

Activation of progelatinase A by mammalian legumain, a recently discovered cysteine proteinase

The activation of progelatinase A to gelatinase A requires cleavage of an asparaginyl bond to form the N-terminus of the mature enzyme. We have asked whether the activation can be mediated by legumain, the recently discovered lysosomal cysteine proteinase that is specific for hydrolysis of asparaginyl bonds. Addition of purified legumain to the concentrated conditioned medium from HT1080 cell culture that contained both progelatinases A and B caused the conversion of the 72 kDa progelatinase A to the 62 kDa form. The progelatinase B in the medium was unaffected. Incubation of recombinant progelatinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Legumain also activated progelatinase A when it was in complex with TIMP-2. Zymographic analysis and N-terminal sequencing revealed that legumain cleaved the 72 kDa progelatinase A at the bonds between Asn109-Tyr110 or Asn111-Phe112 to produce the 62 kDa mature enzyme, and that further cleavage at Asn430 also occurred to generate a 36 kDa active form. More 62 kDa gelatinase A was detected in cultures of C13 cells that over-expressed legumain than in those of the control HEK293 cells. We conclude that legumain is clearly capable of processing progelatinase A to the active enzyme in vitro and in cultured cells.     

Binding of tissue inhibitor of metalloproteinases 2 to two distinct sites on human 72-kDa gelatinase. Identification of a stabilization site.

We have identified a binding site for tissue inhibitors of metalloproteinases 2 (TIMP-2) on human 72-kDa gelatinase that is distinct from the active site. 72-kDa progelatinase is found in a complex with TIMP-2 in the medium of cultured cells and can be activated with organomercurial compounds to yield a gelatinolytic proteinase that remains bound to TIMP-2. Removal of TIMP-2 from 72-kDa progelatinase by reverse-phase high performance liquid chromatography, followed by reconstitution of the progelatinase in neutral pH buffer, results in autocatalytic activation. When samples of autoactivated gelatinase were blotted onto nitrocellulose, then probed with 125I-TIMP-2, we found a 29-kDa peptide that was capable of binding TIMP-2. We isolated this fragment and identified it as the region of gelatinase from amino acid 414 to the carboxyl terminus in the primary amino acid sequence of progelatinase. This portion of the molecule does not contain the putative zinc- or gelatin-binding sites and is proteolytically inactive. Incubation of 125I-TIMP-2 with 72-kDa progelatinase-TIMP-2 complexes resulted in a concentration-dependent exchange of labeled TIMP-2 with unlabeled TIMP-2, in both the presence and absence of the metalloproteinase inhibitor 1,10-phenanthroline. Saturation binding kinetics for the active site of 72-kDa gelatinase were measured in pools of the 43-kDa active fragment that results from the autoactivation of 72-kDa progelatinase; this fragment has no carboxyl-terminal TIMP-2 binding capability. Binding of 125I-TIMP-2 to the active site was completely inhibited by 1,10-phenanthroline. Binding kinetics for the putative stabilization site were determined with isolated 72-kDa progelatinase. In the presence of 1,10-phenanthroline, 72-kDa progelatinase bound 125I-TIMP-2 but not 125I-TIMP-1. Scatchard analysis yielded an approximate dissociation constant (Kd) of 0.72 nM for the active site and 0.42 nM for the stabilization site.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.

Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo r?le of gelatinases is discussed.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.     

Role of Ca2+-dependent metalloprotease-2 in stimulating Ca2+ ATPase activity under peroxynitrite treatment in bovine pulmonary artery smooth muscle membrane

We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO- -triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gelatin-containing zymogram. The 72-kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease-2 (TIMP-2), indicating that the protease is the matrix metalloprotease-2 (MMP-2). Treatment of the membrane suspension with ONOO- caused stimulation of the MMP-2 activity (as evidenced by 14C-gelatin degradation) and also increased Ca2+ ATPase activity. The ONOO- -triggered protease activity and the Ca2+ ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO- -stimulated MMP-2 activity and Ca2+ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO-. Under both basal and ONOO- triggered conditions, the MMP-2 activity and the Ca2+ ATPase activity were also inhibited by EGTA, 1:10-phenanthroline, and TIMP-2. However, the ONOO- -stimulated MMP-2 activity and the Ca2+ ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman-Birk inhibitor, chymostatin, leupeptin, antipain, N-ethylmaleimide, and pepstatin. These results suggest that ONOO- caused stimulation of MMP-2 activity and that the increased MMP-2 activity subsequently played a pivotal role in stimulating Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane.     

Reactive site-modified tissue inhibitor of metalloproteinases-2 inhibits the cell-mediated activation of progelatinase A

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A. To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A. Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the alpha-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate form of gelatinase A to its mature form.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties.

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).     

Domain interactions in the gelatinase A.TIMP-2.MT1-MMP activation complex. The ectodomain of the 44-kDa form of membrane type-1 matrix metalloproteinase does not modulate gelatinase A activation

On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly(285)-Cys(508)), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.     

Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.

The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.     

The sensitivity of versican from rabbit lung to gelatinase A (MMP-2) and B (MMP-9) and its involvement in the development of hydraulic lung edema.

Large chondroitinsulphate-containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP-9 and proMMP-2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP-2 and MMP-9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.     

Complex role of matrix metalloproteinases in angiogenesis

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a significant role in regulating angiogenesis,the process of new blood vessel formation.Interstitial collagenase (MMP-1),72kDa gelatinase A/type IV collagenase (MMP-2),and 92 kDA gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and promote angiogenesis.TIMP-1,TIMP-2,TIMP-3,and possibly,TIMP-4 inhibit neovascularization.A new paradign is emerging that matrilysin (MMP-7),MMP-9,and metalloelastase (MMP-12) may block angiogenesis by converting plasminogen to angiostatin,which is one of the most potent angiogenesis antagonists.MMPs and TIMPs play a complex role in regulating angiogenesis.An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide important information to allow the control of angiogenesis,e.g.the stimulation of angiogenesis for coronary collateral circulation formation;while the inhibition for treating arthritis and cancer.     

Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins

Culture of human dermal fibroblasts within a three-dimensional matrix composed of native type I collagen fibrils is widely used to study the cellular responses to the extracellular matrix. Upon contact with native type I collagen fibrils human skin fibroblasts activate latent 72-kDa type IV collagenase/ gelatinase (MMP-2) to its active 59- and 62-kDa forms. This activation did not occur when cells were cultured on plastic dishes coated with monomeric type I collagen or its denatured form, gelatin. Activation could be inhibited by antibodies against MT1-MMP, by the addition of TIMP-2 and by prevention of MT1-MMP processing. MT1-MMP protein was detected at low levels as active protein in fibroblasts cultured as monolayers. In collagen gel cultures, an increase of the active, 60-kDa MT1-MMP and an additional 63-kDa protein corresponding to inactive MT1-MMP was detected. Incubation of medium containing latent MMP-2 with cell membranes isolated from fibroblasts grown in collagen gels caused activation of the enzyme. Furthermore, regulation of MT1-MMP expression in collagen cultures seems to be mediated by alpha2beta1 integrins. These studies suggest that activation of the proMMP-2 is regulated at the cell surface by a mechanism which is sensitive to cell culture in contact with physiologically relevant matrices and which depends on the ratio of proenzyme and the specific inhibitor TIMP-2.