©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Preferential Inactivation of Tissue Inhibitor of Metalloproteinases-1 That Is Bound to the Precursor of Matrix Metalloproteinase 9 (Progelatinase B) by Human Neutrophil Elastase (*)

Yoshifumi Itoh , Hideaki Nagase (§)

From the (1)Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-9TIMP-1 complex activable by MMP-3 as well as activating pro-MMP-3.


INTRODUCTION

Matrix metalloproteinase 9 (MMP-9),()also designated gelatinase B, is a member of the matrixin family(1) . The enzyme readily digests heat-denatured collagen (gelatins), but it also degrades collagen types IV, V, XI, laminin, elastin (see Refs. 1 and 2 for review), entactin(3) , aggrecan core protein(4) , and cartilage link protein(5) . The enzyme was first found in neutrophils (6) and shown to be immunologically identical to the gelatinase in macrophages (7-9). Recent studies, however, have demonstrated that MMP-9 is also produced in articular chondrocytes(10, 11, 12) , synovial fibroblasts(13) , T-lymphocytes(14, 15) , HT-1080 fibrosarcoma cells, monocytic leukemia cell lines (16) when they are stimulated with interleukin-1, tumor necrosis factor , and/or a phorbol ester. Elevated expression of MMP-9 in cytotrophoblasts(17) , osteoclasts in developing embryos(18) , osteoarthritic chondrocytes(12) , macrophages in rheumatoid synovium (19), and in invasive cancer cells (see Ref. 20 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.

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 HEXXHXXGXXH motif, three repeats of fibronectin type II-like domain and type V collagen-like domain, and a C-terminal hemopexin/vitronectin-like domain(1, 2) . 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)(21, 22, 23) . However, the recent finding that pro-MMP-9 forms a specific complex with an endogenous MMP inhibitor, TIMP-1(16) , 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(22, 24) , 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.()

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(25) , suggests that HNE plays a key role in tissue destruction in inflammatory sites in vivo.


EXPERIMENTAL PROCEDURES

Materials

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(25) . Human pro-MMP-1 (interstitial collagenase) was purified from the medium of the TPA-treated U-937 cells according to Suzuki et al.(26) . 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(27) . Antiserum against human pro-MMP-9 and reduced TIMP-1 were raised in sheep using purified antigens as described previously(28) . Both antisera were shown to be specific by Western blotting analysis using the concentrated crude culture medium of HT-1080 cells.

Cell Cultures

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-9TIMP-1 complex.

Purification of Pro-MMP-9TIMP-1 Complex

Pro-MMP-9TIMP-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 CaCl, 0.02% NaN). 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-9TIMP-1 complex.

Enzyme Assays

All enzyme assays were carried out in TNC buffer containing 0.05% Brij 35. The collagenolytic activity of MMP-1 was measured using C-acetylated type I collagen (guinea pig) according to the method of Cawston and Barret(29) . The gelatinolytic activity of MMP-9 was measured using heat-denatured C-acetylated type I collagen (guinea pig)(30) . One unit of collagenolytic and gelatinolytic activity degraded 1 µg of collagen or gelatin per min at 37 °C.

TIMP Assay

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 C-acetylated type I collagen (guinea pig).

Determination of Concentrations of Pro-MMP-9TIMP-1 Complex, MMP-1, and MMP-3

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-9TIMP-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 and Zymography

Western blotting analysis was carried out as described previously(31) . 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(27) . Enzymic activity was visualized as negative staining with Coomassie Brilliant Blue R-250.


RESULTS

Inactivation of TIMP-1 in the Pro-MMP-9TIMP-1 Complex by HNE

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-9TIMP-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-9TIMP-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 kDa, identical to those of TIMP-1 treated with HNE. The degradation rates of TIMP-1 in the complex and that of free TIMP-1 were almost identical. Although the treatment with trypsin and chymotrypsin also diminished the TIMP-1 activity of the complex, Western blotting analyses indicated that the TIMP-1 molecule was intact. Instead, pro-MMP-9 was processed into lower molecular weight species. TIMP-1, when not bound to pro-MMP-9, is susceptible to degradation by trypsin or chymotrypsin(32) , but both enzymes activate pro-MMP-9(23, 27) . Thus, the loss of MMP inhibitory activity of TIMP-1 in the complex after trypsin or chymotrypsin treatment is likely to result from the formation of the complex through the inhibitory site of TIMP-1 and active site of activated MMP-9, so that the TIMP-1 molecule is no longer available to inhibit other MMPs (MMP-1 in Fig. 1). To verify this, the trypsin or chymotrypsin-treated complex was applied to an anti-TIMP-1 affinity column. The complex, after treatment with trypsin (10 µg/ml) for 1 h at 37 °C, exhibited no TIMP activity, whereas the complex, after treatment with chymotrypsin (10 µg/ml) for 4 h, retained about 34% TIMP activity. However, no gelatinolytic activity was detected in either case (data not shown). Application of these samples to the anti-(TIMP-1) IgG-Affi-Gel 10 column indicated that all low molecular weight species of MMP-9 generated by trypsin or chymotrypsin bound to the column and eluted with 6 M urea together with TIMP-1 (Fig. 3). These results indicate that the activated MMP-9 formed a complex with TIMP-1 through the catalytic site of the enzyme and the N-terminal inhibitory domain of the inhibitor. Plasmin and cathepsin G had very little effect on TIMP-1 and pro-MMP-9.


Figure 1: Inactivation of TIMP-1 in the pro-MMP-9TIMP-1 complex by proteinases. Purified pro-MMP-9TIMP-1 complex (20 µg/ml) was incubated with HNE (5 µg/ml) (), bovine chymotrypsin (10 µg/ml) (), bovine trypsin (10 µg/ml) (), human plasmin (10 µg/ml) (), or human cathepsin G (10 µg/ml) () at 37 °C for indicated periods of time. After incubation, the reactions were terminated with 2 mM Dip-F, and the samples were subjected to the assay for TIMP activity to inhibit the purified 41-kDa MMP-1 as described under ``Experimental Procedures.''




Figure 2: Western blotting analysis of the pro-MMP-9TIMP-1 complex treated with proteinases. The samples from Fig. 1 was analyzed by Western blotting using sheep anti-(human MMP-9) and sheep anti-(human TIMP-1) antibodies as a first antibody. C, pro-MMP-9TIMP-1 complex without treatment; CT, chymotrypsin.




Figure 3: Complex formation between the trypsin- or chymotrypsin-activated MMP-9 and TIMP-1. Pro-MMP-9-TIMP-1 complex (25 µg/ml) was treated with trypsin (10 µg/ml) or chymotrypsin (CT) (10 µg/ml) at 37 °C for 1 or 4 h, respectively. After terminating the reaction with 2 mM Dip-F, the sample (100 µl) was applied to an anti-(TIMP-1)IgG-Affi-Gel 10 column (1 ml) equilibrated with TNC buffer. The unbound materials were collected into a 2-ml fraction, and the bound materials were eluted with TNC buffer comtaining 6 M urea into the same volume. Then 1 ml of each fraction was precipitated with 4.5% trichloroacetic acid with 5 µg of human fibronectin as a carrier. The precipitates were then dissolved in 30 µl of loading buffer containing -mercaptoethanol and applied to Western blotting analysis using sheep anti-(human MMP-9) and anti-(human TIMP-1) antibodies. S, starting materials; U, unbound fraction; B, bound fraction.



Activation of Pro-MMP-9 in the HNE-treated Complex by MMP-3

Incubation of the pro-MMP-9TIMP-1 complex and MMP-3 at a molar ratio of 1:0.2 or 1:0.5 at 37 °C failed to activate pro-MMP-9 in the complex even after 24 h (). This was due to the inhibition of MMP-3 by TIMP-1 of the complex. A very small amount of pro-MMP-9 was converted to an intermediate 86-kDa form after incubation with a 0.5 molar ratio of MMP-3 for 24 h (Fig. 4). This species is an initial product of MMP-9 generated by MMP-3, but it does not have enzymic activity(21) . When the HNE-treated complex was incubated with a catalytic amount of MMP-3, pro-MMP-9 was activated in a time- and a dose-dependent manner. The level of pro-MMP-9 activation was dependent on the degree of TIMP-1 degradation by HNE (, Fig. 4). After a 4-h treatment with HNE, about 30% of TIMP-1 activity was left (Fig. 1). Under these conditions, the addition of a 0.2 molar ratio of MMP-3 to the complex did not activate pro-MMP-9, but a 0.5 molar ratio proportion of MMP-3 did activate the zymogen generating the fully active 82-kDa form (Fig. 4). The lack of activation at a 0.2 molar ratio of MMP-3 can be attributed to the inhibition of MMP-3 by the intact TIMP-1 that was present in the complex. After incubation of the complex with HNE for 8 h, residual TIMP-1 activity was 15% of the original, and a 0.2 molar amount of MMP-3 was able to activate the complex. The maximal activity of MMP-9 detected was about 75% of that generated by treatment of the complex with HNE for 4 or 8 h. A decrease in MMP-9 activity after incubation with a 0.5 molar ratio of MMP-3 at 37 °C for a longer incubation time was due to degradation of MMP-9 either by autolysis or by MMP-3.


Figure 4: Activation of the HNE-treated pro-MMP-9TIMP-1 complex by MMP-3. The same samples in Table I were applied to zymography. Pro-MMP-9 and MMP-9 bands were visualized by negative staining with Coomassie Brilliant Blue R-250.




DISCUSSION

Pro-MMP-9 is activated in vitro by treatment with mercurial compounds, SDS as demonstrated by SDS-containing gelatin zymography, and trypsin. Morodomi et al.(27) reported that pro-MMP-9 can be activated by a relatively high concentration (10 µg/ml) of cathepsin G and plasmin, but the levels of activation were 22-30% and 10%, respectively, while Okada et al.(23) reported more effective activation by these enzymes. Nonetheless, the most likely activator of pro-MMP-9 in vivo is MMP-3 since a catalytic amount of MMP-3 is sufficient to activate this zymogen (21-23). MMP-3 processed the propeptide of pro-MMP-9 in a stepwise manner by initially cleaving the Glu-Met bond and then the Arg-Phe bond for complete removal of the propeptide(21) . However, this action of MMP-3 is readily inhibited when pro-MMP-9 binds to TIMP-1(22) . This is evident from the relatively high affinity between TIMP-1 and MMP-3 as shown by the K value of 0.1 nM and k value of 1.9 10M s(33) . Pro-MMP-9 was found as a complex in the conditioned medium of HT-1080 cells, U937 cells, H-Ras-transformed human fibroblasts, and human alveolar macrophages treated with lipopolysaccharide(34) . When these complexes are treated with APMA or trypsin, pro-MMP-9 in the complex is processed to an active species, but it is readily inhibited by TIMP-1 in the complex. MMP-3 is unable to activate pro-MMP-9 in the complex unless TIMP-1 is saturated with an active MMP.

TIMP-1, on the other hand, can be proteolytically inactivated by a number of non-target proteinases such as HNE and trypsin(32) . In this report, we have demonstrated that HNE preferentially cleaves TIMP-1 and inactivates the inhibitor without affecting the integrity of pro-MMP-9 significantly. The action of HNE on TIMP-1 in the complex is probably on the Val-Cys bond()in the N-terminal domain of the inhibitor as SDS-PAGE analysis of the HNE-treated complex and free TIMP-1 showed fragments with the same molecular masses. After treatment of the complex with the HNE, a catalytic amount of MMP-3 activated pro-MMP-9. After a longer incubation time (e.g. 24 h), less activity of MMP-9 was detected. This resulted from the degradation of the activated MMP-9, possibly by MMP-3. A similar observation was reported recently by Shapiro et al.(35) .

Although trypsin inactivates TIMP-1 by degrading it into several fragments (data not shown), it did not destroy TIMP-1 in the pro-MMP-9TIMP-1 complex (Fig. 2). Under the experimental conditions that we employed, trypsin preferentially activates the zymogen, which in turn binds to the inhibitory site of TIMP-1. It is notable that TIMP-1, in this complex, is resistant to proteolysis by trypsin, suggesting that the trypsin cleavage sites in TIMP-1 are protected by MMP-9.

In conclusion, we have demonstrated that HNE preferentially inactivates TIMP-1 in the pro-MMP-9TIMP-1 complex and renders pro-MMP-9 activable by MMP-3. Shifts of the balance between proteinases and proteinase inhibitors produced by the inactivation of proteinase inhibitors by non-target enzymes are an important consideration in tissue destruction. Examples of this are described for a number of serpins, which are inactivated by bacterial proteinases(36, 37) , cysteine proteinases(38) , and MMPs(39) . The present study suggests that HNE may play an important role in connective tissue destruction under inflammatory conditions not only by its direct action on connective tissue matrix components but also by converting pro-MMP-3 to active MMP-3 (25) and rendering pro-MMP-9 activable by the selective destruction of TIMP-1. The activated MMPs through these mechanisms may further accelerate tissue damage by their direct action on the matrix as well as inactivation of the endogenous elastase inhibitor, -proteinase inhibitor(39) .

  
Table: Expression of gelatinolytic activity in the pro-MMP-9 TIMP-1 complex treated with HNE by catalytic amount of MMP-3

The complex (20 µg/ml) was treated with HNE (5 µg/ml) for indicated periods. After inactivation of HNE by 2 mM Dip-F, the samples were incubated with 0.2 or 0.5 molar ratio of MMP-3 at 37 °C for indicated periods of time. MMP-9 activity was measured using C-labeled guinea pig type I gelatin.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR 39189 and AR 40994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421. Tel.: 913-588-7079; Fax: 913-588-7440.

The abbreviations used are: MMP, matrix metalloproteinase; APMA, 4-aminophenylmercuric acetate; TIMP, tissue inhibitor of metalloproteinases; HNE, human neutrophil elastase; FCS, fetal calf serum; Dip-F, diisopropyl phosphorofluoridate; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hanks' balanced salt solution; PAGE, polyacrylamide gel electrophoresis.

Ogata, Y., Itoh, Y., and Nagase, H. (1995) J. Biol. Chem.270, in press.

H. Nagase, T. E. Cawston, and K. Brew, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Keith Brew for critical reading of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.