Preferential Inactivation of Tissue Inhibitor of Metalloproteinases-1 That Is Bound to the Precursor of Matrix Metalloproteinase 9 (Progelatinase B) by Human Neutrophil Elastase

The precursor of matrix metalloproteinase 9 (pro-MMP-9) forms a complex with the tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule, and the N-terminal domain of TIMP-1 in the complex interacts and inhibits active MMPs. We have reported that a catalytic amount of MMP-3 (stromelysin 1) activates pro-MMP-9 (Ogata, Y., Enghild, J. J., and Nagase, H.(1992) J. Biol. Chem. 267, 3581-3584). To activate pro-MMP-9 in the complex, however, an excess molar amount of MMP-3 is required to saturate the TIMP-1 in the complex. The aim of this study was to test the hypothesis that the requirement for excess MMP-3 can be circumvented by specific destruction of TIMP-1 by non-target proteinases. We have tested trypsin, plasmin, cathepsin G, neutrophil elastase, and chymotrypsin as possible inactivators of TIMP-1 and found that neutrophil elastase inactivates TIMP-1 in the complex without significant destruction of pro-MMP-9. Once TIMP-1 is inactivated, pro-MMP-9 can be readily activated by a catalytic amount of MMP-3. These results suggest that neutrophil elastase may participate in connective tissue destruction at the inflammatory sites not only by its direct action on matrix macromolecules but also by rendering pro-MMP-9 in the pro-MMP-9·TIMP-1 complex activable by MMP-3 as well as activating pro-MMP-3. The precursor of matrix metalloproteinase 9 (pro-MMP-9) forms a complex with the tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule, and the N-terminal domain of TIMP-1 in the complex interacts and inhibits active MMPs. We have reported that a catalytic amount of MMP-3 (stromelysin 1) activates pro-MMP-9 (Ogata, Y., Enghild, J. J., and Nagase, H.(1992) J. Biol. Chem. 267, 3581-3584). To activate pro-MMP-9 in the complex, however, an excess molar amount of MMP-3 is required to saturate the TIMP-1 in the complex. The aim of this study was to test the hypothesis that the requirement for excess MMP-3 can be circumvented by specific destruction of TIMP-1 by non-target proteinases. We have tested trypsin, plasmin, cathepsin G, neutrophil elastase, and chymotrypsin as possible inactivators of TIMP-1 and found that neutrophil elastase inactivates TIMP-1 in the complex without significant destruction of pro-MMP-9. Once TIMP-1 is inactivated, pro-MMP-9 can be readily activated by a catalytic amount of MMP-3. These results suggest that neutrophil elastase may participate in connective tissue destruction at the inflammatory sites not only by its direct action on matrix macromolecules but also by rendering pro-MMP-9 in the pro-MMP-9·TIMP-1 complex activable by MMP-3 as well as activating pro-MMP-3. Matrix metalloproteinase 9 (MMP-9),1 1The abbreviations used are: MMPmatrix metalloproteinaseAPMA4-aminophenylmercuric acetateTIMPtissue inhibitor of metalloproteinasesHNEhuman neutrophil elastaseFCSfetal calf serumDip-Fdiisopropyl phosphorofluoridateTPA12-O-tetradecanoylphorbol-13-acetateDMEMDulbecco's modified Eagle's mediumHBSSHanks' balanced salt solutionPAGEpolyacrylamide gel electrophoresis. also designated gelatinase B, is a member of the matrixin family(1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3091) Google Scholar). The enzyme readily digests heat-denatured collagen (gelatins), but it also degrades collagen types IV, V, XI, laminin, elastin (see Refs. 1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3091) Google Scholar and 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2647) Google Scholar for review), entactin(3Sires U.I. Griffin G.L. Broekelmann T.J. Mecham R.P. Murphy G. Chung A.E. Welgus H.G. Senior R.M. J. Biol. Chem. 1993; 268: 2069-2074Abstract Full Text PDF PubMed Google Scholar), aggrecan core protein(4Fosang A.J. Neame P.J. Last K. Hardingham T.E. Murphy G. Hamilton J.A. J. Biol. Chem. 1992; 267: 19470-19474Abstract Full Text PDF PubMed Google Scholar), and cartilage link protein(5Nguyen Q. Murphy G. Hughes C.E. Mort J.S. Roughley P.J. Biochem. J. 1993; 295: 595-598Crossref PubMed Scopus (95) Google Scholar). The enzyme was first found in neutrophils (6Sopata I. Dancewicz A.M. Biochim. Biophys. Acta. 1974; 370: 510-523Crossref PubMed Scopus (94) Google Scholar) and shown to be immunologically identical to the gelatinase in macrophages (7Mainardi C.L. Hibbs M.S. Hasty K.A. Seyer J.M. Collagen Relat. Res. 1984; 4: 479-492Crossref PubMed Scopus (67) Google Scholar, 8Hibbs M.S. Hoidal J.R. Kang A.H. 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Acta. 1993; 1176: 265-268Crossref PubMed Scopus (70) Google Scholar, 15Zhou H. Bernhard E.J. Fox F.E. Billings P.C. Biochim. Biophys. Acta. 1993; 1177: 174-178Crossref PubMed Scopus (46) Google Scholar), HT-1080 fibrosarcoma cells, monocytic leukemia cell lines (16Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar) when they are stimulated with interleukin-1, tumor necrosis factor α, and/or a phorbol ester. Elevated expression of MMP-9 in cytotrophoblasts(17Librach C.L. Werb Z. Fitzgerald M.L. Chiu K. Corwin N.M. Esteves R.A. Grobelny D. Galardy R. Damsky C.H. Fisher S.J. J. Cell Biol. 1991; 113: 437-449Crossref PubMed Scopus (647) Google Scholar), osteoclasts in developing embryos(18Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Crossref PubMed Scopus (250) Google Scholar), osteoarthritic chondrocytes(12Mohtai M. Smith R.L. Schurman D.J. Tsuji Y. Torti E.M. Huchinson N.I. Stetler-Stevenson W.G. Goldberg G.I. J. Clin. Invest. 1993; 92: 179-185Crossref PubMed Scopus (143) Google Scholar), macrophages in rheumatoid synovium (19Tetlow L.C. Lees M. Ogata Y. Nagase H. Woolley D.E. Rheumatol. Int. 1993; 13: 53-59Crossref PubMed Scopus (69) Google Scholar), and in invasive cancer cells (see Ref. 20Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1527) Google Scholar for review) suggests that the enzyme may play an important role in cellular migration, invasion, and tissue remodeling and catabolism under certain physiological and pathological conditions. matrix metalloproteinase 4-aminophenylmercuric acetate tissue inhibitor of metalloproteinases human neutrophil elastase fetal calf serum diisopropyl phosphorofluoridate 12-O-tetradecanoylphorbol-13-acetate Dulbecco's modified Eagle's medium Hanks' balanced salt solution polyacrylamide gel electrophoresis. MMP-9, like other MMPs, is secreted from cells as an inactive zymogen (pro-MMP-9). Thus, activation of pro-MMP-9 is one of the key steps involved in control of its enzymic activity in the extracellular space. Pro-MMP-9 consists of a propeptide domain, a catalytic domain that contains the zinc-binding HEXXHXXGX XH motif, three repeats of fibronectin type II-like domain and type V collagen-like domain, and a C-terminal hemopexin/vitronectin-like domain(1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3091) Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2647) Google Scholar). The treatment of pro-MMP-9 with trypsin and 4-aminophenylmercuric acetate (APMA) activates the zymogen, but a most likely candidate of pro-MMP-9 activator in vivo is thought to be MMP-3 (stromelysin 1)(21Ogata Y. Enghild J.J. Nagase H. J. Biol. Chem. 1992; 267: 3581-3584Abstract Full Text PDF PubMed Google Scholar, 22Goldberg G.I. Strongin A. Collier I.E. Genrich L.T. Marmer B.L. J. Biol. Chem. 1992; 267: 4519-4583Google Scholar, 23Okada Y. Gonoji Y. Naka K. Tomita K. Nakanishi I. Iwata K. Yamashita K. Hayakawa T. J. Biol. Chem. 1992; 267: 21712-21719Abstract Full Text PDF PubMed Google Scholar). However, the recent finding that pro-MMP-9 forms a specific complex with an endogenous MMP inhibitor, TIMP-1(16Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar), has introduced complexity in the activation of pro-MMP-9 by MMP-3. TIMP-1 binds non-covalently to pro-MMP-9 through the C-terminal domains of the two molecules(22Goldberg G.I. Strongin A. Collier I.E. Genrich L.T. Marmer B.L. J. Biol. Chem. 1992; 267: 4519-4583Google Scholar, 24Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (288) Google Scholar), and the N-terminal domain, which has inhibitory activity, is exposed for interaction with other active MMPs. Therefore, the activation of pro-MMP-9 in the complex by MMP-3 requires more than a molar stoichiometric amount of MMP-3 or blockage of the TIMP-1 by other MMPs.2 2Ogata, Y., Itoh, Y., and Nagase, H. (1995) J. Biol. Chem.270, in press. In this communication, we report another possible mechanism by which pro-MMP-9 in the complex may become readily activable by MMP-3. This results from a specific inactivation of TIMP-1 in the complex by human neutrophil elastase (HNE). This observation, together with the ability of HNE to activate MMP-3 from its precursor(25Nagase H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Crossref PubMed Scopus (351) Google Scholar), suggests that HNE plays a key role in tissue destruction in inflammatory sites in vivo. APMA, Brij 35, diisopropyl phosphorofluoridate (Dip-F), 12-O-tetradecanoylphorbol-13-acetate (TPA), trypsin (bovine), chymotrypsin (bovine), plasminogen (human), urokinase (human), and alkaline phosphatase-conjugated donkey anti-(sheep IgG) IgG were from Sigma. Dulbecco's modified Eagle's medium (DMEM), antibiotics, fetal calf serum (FCS), Hanks' balanced salt solution (HBSS), and lactalbumin hydrolysate were from Life Technologies, Inc. Human fibrosarcoma cell line HT-1080 was obtained from American Type Culture Collection. HNE and human neutrophil cathepsin G were from Athens Research Technology Inc. Human pro-MMP-3 was purified from the culture medium of rheumatoid synovial fibroblasts and activated as described previously(25Nagase H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Crossref PubMed Scopus (351) Google Scholar). Human pro-MMP-1 (interstitial collagenase) was purified from the medium of the TPA-treated U-937 cells according to Suzuki et al.(26Suzuki K. Enghild J.J. Morodomi T. Salvesen G. Nagase H. Biochemistry. 1990; 29: 10261-10270Crossref PubMed Scopus (385) Google Scholar). Pro-MMP-1 was activated with plasmin and MMP-3, and MMP-3 and plasmin were removed by chromatography on anti-MMP-3 IgG coupled to Affi-Gel 10 and Sephacryl S-200, respectively. TIMP-1 was purified from the medium of U-937 cells(27Morodomi T. Ogata Y. Sasaguri Y. Morimatsu M. Nagase H. Biochem. J. 1992; 285: 603-611Crossref PubMed Scopus (179) Google Scholar). Antiserum against human pro-MMP-9 and reduced TIMP-1 were raised in sheep using purified antigens as described previously(28Okada Y. Takeuchi N. Tomita K. Nakanishi I. Nagase H. Ann. Rheum. Dis. 1989; 48: 645-653Crossref PubMed Scopus (174) Google Scholar). Both antisera were shown to be specific by Western blotting analysis using the concentrated crude culture medium of HT-1080 cells. HT-1080 cells were cultured in DMEM containing 10% FCS. After confluency the cells were washed with HBSS and treated with TPA (20 ng/ml) in serum-free DMEM supplemented with 0.2% lactalbumin hydrolysate for 2 days. This conditioned medium was harvested and used for purification of the pro-MMP-9·TIMP-1 complex. Pro-MMP-9·TIMP-1 complex was purified from conditioned medium of HT-1080 cells. First, culture medium was passed through a column of gelatin-Sepharose 4B equilibrated with TNC buffer (50 mM Tris-HCl (pH. 7.5), 0.15 M NaCl, 10 mM CaCl2, 0.02% NaN3). The column was washed with the same buffer, and the enzyme was subsequently eluted with the same buffer containing 2% dimethyl sulfoxide. The eluted protein was pooled, dialyzed against TNC buffer, concentrated with an Amicon YM-10 membrane, and subjected to gel permeation chromatography on Sephacryl S-300. The protein peak containing pro-MMP-9 was mostly a complex with TIMP-1. Fractions from the front edge of the peak were pooled as the pro-MMP-9·TIMP-1 complex. All enzyme assays were carried out in TNC buffer containing 0.05% Brij 35. The collagenolytic activity of MMP-1 was measured using 14C-acetylated type I collagen (guinea pig) according to the method of Cawston and Barret(29Cawston T.E. Barrett A.J. Anal. Biochem. 1979; 99: 340-345Crossref PubMed Scopus (268) Google Scholar). The gelatinolytic activity of MMP-9 was measured using heat-denatured 14C-acetylated type I collagen (guinea pig)(30Harris Jr., E.D. Krane S.M. Biochim. Biophys. Acta. 1972; 258: 566-576Crossref PubMed Scopus (159) Google Scholar). One unit of collagenolytic and gelatinolytic activity degraded 1 μg of collagen or gelatin per min at 37°C. TIMP activity was measured against the 41-kDa MMP-1. Various concentrations of the samples were incubated with a constant amount of MMP-1 at 37°C for 30 min, and residual collagenolytic activity was measured against 14C-acetylated type I collagen (guinea pig). The amount of the 41-kDa MMP-1 and the 45-kDa MMP-3 was determined by titration with TIMP-1. The amount of the pro-MMP-9·TIMP-1 complex was determined by titration of the TIMP-1 in the complex with MMP-1 or MMP-3, assuming the molar ratio of pro-MMP-9 to TIMP-1 in the complex is 1:1. Western blotting analysis was carried out as described previously(31Ito A. Nagase H. Arch. Biochem. Biophys. 1988; 267: 211-216Crossref PubMed Scopus (108) Google Scholar). Sheep anti-(human MMP-9) IgG was used as a primary antibody at a concentration of 5 μg/ml and sheep anti-(human TIMP-1) serum at a 1:1000 dilution, and alkaline phosphatase-conjugated donkey anti-(sheep IgG) IgG was used for a secondary antibody. Zymography was conducted with SDS-polyacrylamide gel containing gelatin (0.8 mg/ml) as described previously(27Morodomi T. Ogata Y. Sasaguri Y. Morimatsu M. Nagase H. Biochem. J. 1992; 285: 603-611Crossref PubMed Scopus (179) Google Scholar). Enzymic activity was visualized as negative staining with Coomassie Brilliant Blue R-250. Five different proteinases (bovine trypsin, bovine chymotrypsin, HNE, human cathepsin G, and human plasmin) were tested for their ability to inactivate the TIMP-1 component of the pro-MMP-9·TIMP-1 complex. The treatment of the complex with trypsin, HNE, and chymotrypsin diminished apparent TIMP-1 activity in a time-dependent manner, but plasmin or cathepsin G had little effect (Fig. 1). SDS-PAGE and immunoblotting analysis of the pro-MMP-9·TIMP-1 complex after treatment with these proteinases showed that only HNE degraded TIMP-1 in a time-dependent manner (Fig. 2). HNE also cleaved some of the pro-MMP-9, but the amount was low in comparison with TIMP-1 degradation. The degradation products of TIMP-1 had molecular masses of 17 and 16 identical to of TIMP-1 treated with The degradation of TIMP-1 in the complex and that of TIMP-1 were the treatment with trypsin and chymotrypsin also diminished the TIMP-1 activity of the complex, Western blotting that the TIMP-1 was pro-MMP-9 was molecular when not to pro-MMP-9, is to degradation by trypsin or Y. S. Nakanishi I. J. Hayakawa T. J. Nagase H. 1989; Scopus Google Scholar), but activate Y. Gonoji Y. Naka K. Tomita K. Nakanishi I. Iwata K. Yamashita K. Hayakawa T. J. Biol. Chem. 1992; 267: 21712-21719Abstract Full Text PDF PubMed Google Scholar, T. Ogata Y. Sasaguri Y. Morimatsu M. Nagase H. Biochem. J. 1992; 285: 603-611Crossref PubMed Scopus (179) Google Scholar). Thus, the of MMP inhibitory activity of TIMP-1 in the complex after trypsin or chymotrypsin treatment is likely to from the of the complex through the inhibitory of TIMP-1 and active of activated MMP-9, that the TIMP-1 is to other in 1). To the trypsin or complex was to an The complex, after treatment with trypsin for 1 at TIMP activity, the complex, after treatment with chymotrypsin for TIMP However, gelatinolytic activity was in not of these samples to the 10 column that low molecular of MMP-9 by trypsin or chymotrypsin to the column and eluted with M together with TIMP-1 (Fig. These results that the activated MMP-9 a complex with TIMP-1 through the catalytic of the enzyme and the N-terminal inhibitory domain of the and cathepsin G had little effect on TIMP-1 and blotting analysis of the pro-MMP-9·TIMP-1 complex treated with proteinases. The samples 1 was by Western blotting using sheep anti-(human MMP-9) and sheep anti-(human TIMP-1) as a first antibody. pro-MMP-9·TIMP-1 complex without the or MMP-9 and TIMP-1. complex was treated with trypsin or chymotrypsin at 37°C for 1 or respectively. After the with 2 mM the was to an 10 column equilibrated with TNC The were a and the were eluted with TNC buffer M the same 1 of each was with with 5 μg of human fibronectin as a The were in 30 of buffer containing and to Western blotting analysis using sheep anti-(human MMP-9) and anti-(human TIMP-1) B, of the pro-MMP-9·TIMP-1 complex and MMP-3 at a molar ratio of or at 37°C to activate pro-MMP-9 in the complex after This was to the of MMP-3 by TIMP-1 of the complex. amount of pro-MMP-9 was to an after with a molar ratio of MMP-3 for (Fig. This is an of MMP-9 by MMP-3, but it not have enzymic Y. Enghild J.J. Nagase H. J. Biol. Chem. 1992; 267: 3581-3584Abstract Full Text PDF PubMed Google Scholar). the complex was incubated with a catalytic amount of MMP-3, pro-MMP-9 was activated in a and a The of pro-MMP-9 activation was on the of TIMP-1 degradation by HNE After a treatment with HNE, of TIMP-1 activity was (Fig. 1). these the of a molar ratio of MMP-3 to the complex not activate pro-MMP-9, but a molar ratio of MMP-3 activate the zymogen the active (Fig. The of activation at a molar ratio of MMP-3 can be to the of MMP-3 by the TIMP-1 that was in the complex. After of the complex with HNE for residual TIMP-1 activity was of the and a molar amount of MMP-3 was to activate the complex. The activity of MMP-9 was of that by treatment of the complex with HNE for or in MMP-9 activity after with a molar ratio of MMP-3 at 37°C for a was to degradation of MMP-9 by or by of gelatinolytic activity in the pro-MMP-9 TIMP-1 complex treated with HNE by catalytic amount of MMP-3 in a Pro-MMP-9 is activated in by treatment with as demonstrated by gelatin and Morodomi et T. Ogata Y. Sasaguri Y. Morimatsu M. Nagase H. Biochem. J. 1992; 285: 603-611Crossref PubMed Scopus (179) Google Scholar) reported that pro-MMP-9 can be activated by a concentration of cathepsin G and plasmin, but the of activation were and et Y. Gonoji Y. Naka K. Tomita K. Nakanishi I. Iwata K. Yamashita K. Hayakawa T. J. Biol. Chem. 1992; 267: 21712-21719Abstract Full Text PDF PubMed Google Scholar) reported more activation by these the most likely activator of pro-MMP-9 in vivo is MMP-3 a catalytic amount of MMP-3 is to activate this zymogen MMP-3 the propeptide of pro-MMP-9 in a manner by the and the for of the Y. Enghild J.J. Nagase H. J. Biol. Chem. 1992; 267: 3581-3584Abstract Full Text PDF PubMed Google Scholar). However, this action of MMP-3 is readily when pro-MMP-9 binds to G.I. Strongin A. Collier I.E. Genrich L.T. Marmer B.L. J. Biol. Chem. 1992; 267: 4519-4583Google Scholar). This is from the TIMP-1 and MMP-3 as shown by the of and of Q. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; PubMed Scopus Google Scholar). Pro-MMP-9 was found as a complex in the conditioned medium of HT-1080 cells, cells, human and human macrophages treated with H.G. E.J. Eisen A.Z. Senior R.M. S.M. Goldberg G.I. J. Clin. Invest. 1990; PubMed Scopus Google Scholar). these are treated with or trypsin, pro-MMP-9 in the complex is to an active but it is readily by TIMP-1 in the MMP-3 is to activate pro-MMP-9 in the complex TIMP-1 is with an active on the other can be by a of non-target proteinases as HNE and Y. S. Nakanishi I. J. Hayakawa T. J. Nagase H. 1989; Scopus Google Scholar). In this we have demonstrated that HNE TIMP-1 and inactivates the inhibitor without the of pro-MMP-9 The action of HNE on TIMP-1 in the complex is on the Nagase, T. and K. in in the N-terminal domain of the inhibitor as SDS-PAGE analysis of the complex and TIMP-1 showed with the same molecular After treatment of the complex with the HNE, a catalytic amount of MMP-3 activated pro-MMP-9. After a activity of MMP-9 was This from the degradation of the activated MMP-9, by MMP-3. was reported by et Broekelmann T.J. Mecham R.P. Senior R.M. Welgus H.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). trypsin inactivates TIMP-1 by it not it not TIMP-1 in the pro-MMP-9·TIMP-1 complex (Fig. 2). the that we trypsin activates the zymogen, which in binds to the inhibitory of TIMP-1. is that in this complex, is to by trypsin, that the trypsin sites in TIMP-1 are by In we have demonstrated that HNE inactivates TIMP-1 in the pro-MMP-9·TIMP-1 complex and pro-MMP-9 activable by MMP-3. of the proteinases and produced by the inactivation of by non-target are an important in tissue of this are described for a of which are by K. H. M. Iwata T. J. Biochem. 1984; PubMed Scopus Google Scholar, J. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), D. A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and A.E. Enghild J.J. Nagase H. Suzuki K. Salvesen G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The study suggests that HNE may play an important role in connective tissue destruction under inflammatory not only by its direct action on connective tissue matrix but also by pro-MMP-3 to active MMP-3 H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Crossref PubMed Scopus (351) Google Scholar) and rendering pro-MMP-9 activable by the destruction of TIMP-1. The activated through these may tissue by their direct action on the matrix as well as inactivation of the endogenous elastase inhibitor, A.E. Enghild J.J. Nagase H. Suzuki K. Salvesen G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The complex (20 was treated with HNE for After inactivation of HNE by 2 mM the samples were incubated with or molar ratio of MMP-3 at 37°C for of MMP-9 activity was measured using type I We for of the