©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Steps Involved in Activation of the Pro-matrix Metalloproteinase 9 (Progelatinase B)-Tissue Inhibitor of Metalloproteinases-1 Complex by 4-Aminophenylmercuric Acetate and Proteinases (*)

(Received for publication, March 22, 1995; and in revised form, May 26, 1995)

Yutaka Ogata (§) Yoshifumi Itoh Hideaki Nagase (¶)

From the 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 (proMMP-9, progelatinase B) noncovalently binds to tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule. We have isolated the proMMP-9bulletTIMP-1 complex from the medium of human fibrosarcoma HT-1080 cells and investigated the activation processes of the complex by 4-aminophenylmercuric acetate, trypsin, and matrix metalloproteinase 3 (MMP-3, stromelysin 1). The treatment of the proMMP-9bulletTIMP-1 complex with 4-aminophenylmercuric acetate or trypsin converts proMMP-9 to lower molecular weight species corresponding to active forms, but no gelatinolytic activity is detected. The lack of enzymic activity results from binding of TIMP-1 to the activated MMP-9. The treatment of the proMMP-9bulletTIMP-1 complex with a possible physiological proMMP-9 activator, MMP-3, does not reveal any gelatinolytic activity unless the molar ratio of MMP-3 to the complex exceeds 1. This is due to the inhibition of MMP-3 by TIMP-1 forming a ternary proMMP-9bulletTIMP-1bulletMMP-3 complex. The formation of the ternary complex weakens the interaction between proMMP-9 and TIMP-1, resulting in partial dissociation of the complex into proMMP-9 and the TIMP-1bulletMMP-3 complex. When MMP-3 is in excess, the propeptide is completely processed, and the full activity of MMP-9 is detected. Similarly, the proMMP-9bulletTIMP-1 complex inhibits MMP-1 (interstitial collagenase) and in turn renders the proMMP-9 activable by a catalytic amount of MMP-3. These results suggest that formation of the proMMP-9bulletTIMP-1 complex regulates extracellular matrix breakdown in tissue by switching the predominant MMP activity from one type to another.


INTRODUCTION

Matrix metalloproteinases (MMPs), (^1)also designated as matrixins, are a group of structurally related zinc metalloendopeptidases capable of degrading extracellular matrix components (for review, see (1) and (2) ). These enzymes are considered to play a key role in normal tissue remodeling and in pathological destruction of the matrix in many connective tissue diseases such as arthritis, periodontitis, and tissue ulceration and in cancer cell invasion and metastasis(1, 2, 3) . Activities of the MMPs are precisely controlled, not only by their gene expression in various cell types but also by activation of their precursors (proMMPs) and inhibition by endogenous inhibitors, such as alpha(2)-macroglobulin (alpha(2)M) and tissue inhibitors of metalloproteinases (TIMPs)(1, 2, 3) .

To date, 11 members of the matrixin family have been identified, and their enzymic activities have been characterized(1, 2, 4, 5, 6, 7, 8) . These include three collagenases (interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13)(4) ), two gelatinases (gelatinase A (MMP-2) and gelatinase B (MMP-9)), two stromelysins (stromelysin 1 (MMP-3) and stromelysin 2 (MMP-10)), matrilysin (MMP-7), stromelysin 3 (MMP-11)(5, 6) ), macrophage metalloelastase (MMP-12)(7) , and membrane-type MMP (MMP-14)(8) . Among them, MMP-2 and MMP-9 are unique in that their zymogen forms can form a complex with TIMP-2 and TIMP-1, respectively(9, 10, 11, 12, 13, 14, 15, 16) . The progelatinase-TIMP complexes are formed through the interaction of the C-terminal hemopexin/vitronectin-like domain of the zymogen and the C-terminal domain of the inhibitor(16, 17, 18, 19, 20, 21) . Since the inhibitory activity of TIMPs resides in the N-terminal domain(17, 22) , the proMMP-2bulletTIMP-2 and the proMMP-9bulletTIMP-1 complexes are capable of inhibiting active MMPs(10, 15, 23, 24) . This introduces complexity in activation of progelatinase-TIMP complexes. proMMP-2 free of TIMP-2 can be readily activated by a mercurial compound, 4-aminophenylmercuric acetate (APMA)(25) , and by a specific cell surface activator expressed in fibroblasts and cancer cells treated with concanavalin A or a phorbol ester(26, 27) . Recently, membrane-type MMP has been identified as an activator of proMMP-2(8) ; however, the activation of proMMP-2 by a cell surface activator is prevented in the presence of TIMP-2(28, 29) . The proMMP-9bulletTIMP-1 complex is also resistant to activation by MMP-3(21) , while proMMP-9 free of TIMP-1 is readily activated by MMP-3 (21, 30, 31) . On the other hand, many investigators reported that treatment of the proMMP-2bulletTIMP-2 complex or the proMMP-9bulletTIMP-1 complex with APMA yielded gelatinolytic activity with concomitant conversion of the proenzyme to active forms, although the specific activities of the activated complexes were considerably lower than those of TIMP-free gelatinases(11, 12, 21, 23, 24, 34) . By contrast, our studies on the activation of the proMMP-2bulletTIMP-2 complex by APMA have indicated that the activated proMMP-2 is inhibited by the TIMP-2 before it loses the propeptide by autocatalysis(24) . To attain the enzymic activity, masking of the N-terminal inhibitory domain of TIMP-2 with another active MMP is prerequisite(24) .

In this report, we have isolated the human proMMP-9bulletTIMP-1 and investigated the molecular rearrangements that occur in the complex upon activation with APMA and trypsin. To address a possible biological implication of the proMMP-9bulletTIMP-1 complex formation to extracellular matrix catabolism, we have also investigated the conditions that are required for the activation of the complex by MMP-3, a likely proMMP-9 activator in vivo.


EXPERIMENTAL PROCEDURES

Materials

APMA, Brij 35, diisopropylphosphorofluoridate (DFP), 12-O-tetradecanoylphorbol-13-acetate (TPA), trypsin (bovine), plasminogen (human), urokinase, and alkaline phosphatase-conjugated donkey anti-(sheep IgG) IgG were from Sigma. Dulbecco's modified Eagle's medium, RPMI 1640 medium, antibiotics, fetal calf serum, and lactalbumin hydrolysate were from Life Technologies, Inc. Gelatin-Sepharose 4B, Sephacryl S-200, and S-300 were from Pharmacia Biotech Inc. DE-52 (DEAE-cellulose) was from Whatman. Green A Matrex gel and YM-10 membrane were from Amicon Corp. Affi-Gel 10 was from Bio-Rad. Human alpha(2)M was a gift from Dr. Jan J. Enghild, Duke University Medical Center. Human fibrosarcoma cell line HT-1080 cell was obtained from American Type Culture Collection.

Cell Cultures

HT-1080 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics. After confluency, cells were washed with Hanks' balanced salt solution and treated with TPA (20 ng/ml) in serum-free Dulbecco's modified Eagle's medium supplemented with 0.2% lactalbumin hydrolysate for 2 days. The conditioned medium was harvested and used for purification of the proMMP-9bulletTIMP-1 complex.

Anti-(human TIMP-1) IgG-Sepharose

Polyclonal anti-(human TIMP-1) antibodies were prepared in sheep by intramuscular injection of the purified human TIMP-1 from U-937 cells (35) emulsified with complete Freund's adjuvant once and twice with incomplete Freund's adjuvant as described previously(36) . The antiserum obtained was shown to be specific for human TIMP-1 by Western blotting analysis. Sheep anti-(human TIMP-1) IgG isolated by ammonium sulfate precipitation (40% saturation) and DE-52 fractionation was coupled to Affi-Gel 10 and used as immunoadsorbent beads.

Enzyme Assays

All enzymic assays were carried out in TNC buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM Ca, 0.05% Brij 35, 0.02% NaN(3)). The collagenolytic activity of MMP-1 was measured using ^14C-acetylated type I collagen (guinea pig) according to Cawston and Barrett(37) . The gelatinolytic activity of MMP-9 was measured using heat-denatured ^14C-acetylated type I collagen (guinea pig)(38) . One unit of collagenolytic and gelatinolytic activity degraded 1 µg of collagen or gelatin/min at 37 °C.

Purification of proMMP-9bulletTIMP-1 Complex

The proMMP-9bulletTIMP-1 complex was purified from the culture medium of TPA-treated HT-1080 cells. The medium was applied to a gelatin-Sepharose 4B column equilibrated with 50 mM Tris-HCl (pH 7.5), 0.4 M NaCl, 10 mM CaCl(2), 0.02% NaN(3). The column was washed with the same buffer, and proMMP-9 bound was eluted with same buffer containing 2% (v/v) dimethyl sulfoxide. The fractions were pooled, concentrated with an Amicon YM-10 membrane, and applied to Sephacryl S-300. SDS-PAGE analysis indicated that most of the proMMP-9 eluted from the gelatin-Sepharose was in a complex with TIMP-1. Earlier fractions from the column of Sephacryl S-300 were pooled and used for experiments as the proMMP-9bulletTIMP-1 complex. A possible contamination of free proMMP-9 was examined by applying the purified complex (100 µg/ml, 100 µl) onto a column of anti-(human TIMP-1) IgG/Affi-Gel 10 (0.5 ml) equilibrated with TNC buffer. The unbound fraction (2 ml) and the bound material, eluted with 2 ml of 50 mM Tris-HCl (pH 7.5), 6 M urea, 10 mM CaCl(2), 0.02% NaN(3), were analyzed by SDS-PAGE. The purified complex was essentially free of free proMMP-9.

Purification of MMP-3 and MMP-1

Human proMMP-3 was purified from the culture medium of rheumatoid synoviocytes and activated with chymotrypsin as described previously(39) . Human proMMP-1 was purified from TPA-treated U-937 cell culture medium and activated by plasmin and MMP-3 according to Suzuki et al.(40) . After activation, plasmin was inactivated by 2 mM DFP, and the 41-kDa MMP-1 was isolated by chromatography on anti-MMP-3 immunoadsorbent and on Sephacryl S-200. MMP-1 was free of MMP-3 and plasmin.

Estimation of the Amount of the proMMP-9-TIMP-1 Complex, MMP-1, and MMP-3

The amounts of the 41-kDa MMP-1 and the 45-kDa MMP-3 were estimated by titration with a known amount of TIMP-1. The amount of the proMMP-9bulletTIMP-1 complex was estimated by titration of TIMP-1 in the complex with the known amount of MMP-1 or MMP-3, assuming the molar ratio of proMMP-9 to TIMP-1 of the complex is 1.

Electrophoresis, Zymography, and Western Blotting

SDS-PAGE was performed according to the method of Bury(41) , and proteins were stained with silver nitrate(42) . Zymography was conducted using SDS-polyacrylamide gels containing gelatin (0.8 mg/ml) as described previously(35) . Enzymic activity was visualized as negative staining with Coomassie Brilliant Blue R-250. Western blotting was carried out according to Ito and Nagase(43) . Sheep anti-(human proMMP-9) IgG, sheep anti-(human TIMP-1) serum, sheep anti-(human MMP-3) serum, and/or sheep anti-(human MMP-1) serum were used as primary antibody, and alkaline phosphatase-conjugated donkey anti-(sheep IgG) IgG was used as secondary antibody.


RESULTS

Isolation of the proMMP-9bulletTIMP-1 Complex

The conditioned medium of TPA-stimulated HT-1080 cells contained the proMMP-9bulletTIMP-1 complex as well as a small amount of free proMMP-9 and free TIMP-1. The gelatin-Sepharose step isolated the complex and free proMMP-9. The separation of the complex from free proMMP-9 was attained by gel permeation chromatography on Sephacryl S-300. Application of the final product to an anti-(human TIMP-1) immunoadsorbent column showed that the purified complex was virtually free of free proMMP-9 (Fig. 1). A very small amount of proMMP-9 (<3%) was found in the unbound fraction of the anti-TIMP-1 column (Fig. 1, lane2), but it is not known whether this is due to partial dissociation of the complex or contamination of free proMMP-9. Nevertheless, neither case affects the interpretation of our studies, as the amount of free proMMP-9, if any, is very small.


Figure 1: SDS-PAGE analysis of the proMMP-9bulletTIMP-1 complex. The purified proMMP-9bulletTIMP-1 complex was applied to anti-(TIMP-1) IgG-Sepharose affinity chromatography to examine the presence of free proMMP-9. The samples before and after the chromatography were analyzed by SDS-PAGE under reducing conditions. Lane 1, the purified proMMP-9bulletTIMP-1 complex before anti-(TIMP-1) IgG-Sepharose; lane 2, the fraction unbound to anti-(TIMP-1) IgG-Sepharose; lane 3, the fraction bound to anti-(TIMP-1) IgG-Sepharose. See ``Experimental Procedures'' for details. The gel was stained with silver nitrate.



Lack of Enzymic Activity of the proMMP-9bulletTIMP-1 Complex upon Treatment with APMA or Trypsin

proMMP-9 is activated by APMA and trypsin(21, 31, 32) . In contrast, when the proMMP-9bulletTIMP-1 complex was treated with these agents, the gelatinolytic activity detected was negligible (Table 1). This suggests that TIMP-1 in the complex may inhibit the activated MMP-9 or prevent the activation of proMMP-9. SDS-PAGE analysis of the APMA-treated complex under reducing conditions indicated that proMMP-9 was mostly converted to an 84-kDa species and to a lesser extent an 82-kDa species (Fig. 2A). Zymographic analysis of the samples under nonreducing conditions indicated that only a small portion of the 82- and 80-kDa species showed gelatinolytic activity, suggesting that most of the converted MMP-9 was inhibited (Fig. 2B). In contrast, when proMMP-9 free from TIMP-1 was activated with APMA, the 80-kDa species was further converted to another active form of 68-kDa by an autolytic reaction as demonstrated previously(35) . Absence of the 68-kDa species in the APMA-treated complex further supports the notion that the enzymic activities of the 82- and the 80-kDa species are inhibited by TIMP-1. Treatment of the proMMP-9bulletTIMP-1 complex with trypsin converted proMMP-9 to 82 and 68 kDa and four other smaller fragments (Fig. 3A). The TIMP-1 molecule was intact under these conditions (Fig. 3A). The 82- and 68-kDa species are identical to those generated with free proMMP-9 treated with trypsin(35) . Similar to APMA treatment, trypsin-treated samples exhibited only a low gelatinolytic activity by zymography (Fig. 3B). Since no significant gelatinolytic activity was detected with these samples using ^14C-acetylated gelatin in solution, a small activity detected by zymography might have resulted from partial dissociation of the active MMP-9bulletTIMP-1 complex due to SDS treatment. Indeed, when the APMA- or trypsin-treated complexes were subjected to Western blotting analysis for TIMP-1 under nonreducing conditions, a majority of TIMP-1 was located at around 90-100 kDa (data not shown), suggesting that the activated MMP-9 formed a complex with TIMP-1.




Figure 2: SDS-PAGE and zymographic analyses of the proMMP-9bulletTIMP-1 complex treated with APMA. The complex (10 µg/ml) was treated with 1 mM APMA at 37 °C for the indicated periods of time. The samples were then subjected to SDS-PAGE under reducing conditions (A) and zymography under nonreducing conditions (B).




Figure 3: SDS-PAGE and zymographic analysis of the proMMP-9bulletTIMP-1 complex treated with trypsin. The complex (10 µg/ml) was treated with trypsin (10 µg/ml) at 37 °C for the indicated periods of time. After inactivating trypsin with 2 mM DFP, the samples were subjected to SDS-PAGE under reducing conditions (A) and zymography under nonreducing conditions (B).



The lack of proteolytic activity in the APMA- and the trypsin-activated complexes was further examined for their abilities to bind to alpha(2)M. Only proteolytically active enzymes are able to bind to alpha(2)M and form a large enzyme-inhibitor complex(44) . We previously demonstrated that an active MMP-9 bound to alpha(2)M stoichiometrically, which resulted in the shift of gelatin-lysis zones of activated MMP-9 in zymography(35) . Inability of the enzyme to bind to alpha(2)M is indicative of the lack of proteolytic activity. When the APMA- or trypsin-activated proMMP-9bulletTIMP-1 complex was reacted with a 4-fold molar excess of alpha(2)M at 37 °C for 1 h prior to zymographic analysis, all species exhibiting gelatinolytic activity by zymography failed to bind to alpha(2)M (Fig. 4), indicating that the APMA- or trypsin-activated proMMP-9bulletTIMP-1 complex does not possess proteolytic activity.


Figure 4: Lack of binding of the APMA- or trypsin-treated proMMP-9bulletTIMP-1 complex to alpha(2)M. The proMMP-9bulletTIMP-1 complex (10 µg/ml) was treated with 1 mM APMA for 24 h or trypsin (10 µg/ml) for 30 min at 37 °C. Trypsin was inactivated with 2 mM DFP. The samples were then diluted 10-fold with TNC buffer, reacted with alpha(2)M (500 µg/ml) at 37 °C for 1 h, and applied to zymographic analysis under nonreducing conditions. C, the control sample without activation; APMA, the complex treated with 1 mM APMA for 24 h; Trypsin, the complex treated with 10 µg/ml of trypsin for 30 min.



Loss of MMP Inhibition Activity of the proMMP-9bulletTIMP-1 Complex after APMA or Trypsin Treatment

The proMMP-9bulletTIMP-1 complex was able to inhibit active MMPs. Fig. 5shows the stoichiometric inhibition of MMP-1 (interstitial collagenase) by the complex. When the complex was treated with APMA or trypsin, however, the inhibitory activity of the complex against MMP-1 was completely lost. This indicates that MMP-9 and TIMP-1 form a complex through their reactive sites after activation of proMMP-9 and the inhibitory domain of TIMP-1 is no longer available for other MMPs.


Figure 5: Loss of MMP-1 inhibition activity of the proMMP-9bulletTIMP-1 complex upon treatment with APMA or trypsin. The proMMP-9bulletTIMP-1 complex was treated with APMA or trypsin, and then its ability to inhibit MMP-1 (1.1 pmol) was measured by determining the residual collagenolytic activity of MMP-1. bullet, the complex without treatment; , the complex treated with 1 mM APMA for 24 h at 37 °C; ▪, the complex treated with trypsin (10 µg/ml) for 1 h at 37 °C followed by inactivation of trypsin with 2 mM DFP.



Activation of the proMMP-9bulletTIMP-1 Complex by MMP-3

A catalytic amount of MMP-3 activates proMMP-9 in a stepwise manner(31) ; however, activation of the proMMP-9bulletTIMP-1 complex by MMP-3 is not readily achieved(21) . This is anticipated, as MMP-3 can be readily inhibited by the complex. Therefore, we postulated that activation of the complex might be possible if TIMP-1 present in the complex was saturated with an active MMP. To investigate this, the complex was incubated with an increasing amount of MMP-3 for 1 and 4 h at 37 °C and the MMP-9 activity was measured against ^14C-acetylated gelatin. The gelatinolytic activity of MMP-3 is 0.2% of that of MMP-9 (45) ; thus, the contribution of MMP-3 in this assay system is negligible. As shown in Fig. 6, gelatinolytic activity was observed only when the molar ratio of MMP-3 to the complex exceeded 1. The maximal specific activity of MMP-9 detected by incubation of the complex with MMP-3 at a 1:2 molar ratio for 4 h was 20,000 units/mg, about the same activity of the fully activated MMP-9(31, 35) . Zymographic analysis of each incubation mixture showed that the active 80-kDa (82-kDa with reduction) species was generated only after a molar ratio of MMP-3 to the complex exceeded 1. proMMP-9 was partially processed to 84-kDa (86-kDa with reduction) below a 1:1 molar ratio. This species is not proteolytically active, and the conversion of the 84-kDa species to the active 80-kDa form requires the specific hydrolysis of Arg-Phe by MMP-3(31) . Lack of proteolytic activity of the 84-kDa species as well as proMMP-9 was demonstrated by their inability to bind to alpha(2)M, whereas most of the 80-kDa species bound to alpha(2)M and shifted to the top of the gel where alpha(2)M-proteinase complexes run on SDS-PAGE under nonreducing conditions (Fig. 6B). A small amount of activity remaining at 80-kDa is probably due to partial dissociation of the enzyme from the alpha(2)M complex by SDS treatment(35) .


Figure 6: Activation of the proMMP-9bulletTIMP-1 complex by 45-kDa MMP-3. The complex were incubated with the 45-kDa MMP-3 at various molar ratios at 37 °C for 1 and 4 h. The samples were subjected to measurement of gelatinolytic activity (A) and zymographic analysis (B).



Interaction of the proMMP-9bulletTIMP-1 Complex with MMP-3

The inability of MMP-3 to activate proMMP-9 bound to TIMP-1 when the amount of MMP-3 was below a 1:1 molar stoichiometry suggests that MMP-3 preferentially interacts with TIMP-1. We then examined whether active MMP-3 forms a stable proMMP-9bulletTIMP-1bulletMMP-3 ternary complex. To investigate this, the proMMP-9bulletTIMP-1 complex was reacted with MMP-3 at different molar ratios, and the mixtures were applied to a gelatin-Sepharose column. Since both proMMP-9 and MMP-9 bind to the column, any molecules that are recovered in the bound fraction are associated with either proMMP-9 or MMP-9. As shown in Fig. 7A, TIMP-1 of the complex was recovered in the bound fraction, but MMP-3 was not. When MMP-3 was reacted with the complex at molar ratios to the complex of 0.5:1 and 1:1 at 37 °C and the mixtures were applied to the column immediately or 1 h after the reaction, MMP-3 was recovered in both the unbound and the bound fractions in approximately equal amounts in both cases (Fig. 7B). proMMP-9 recovered in the bound fraction remained primarily as a zymogen. The recovery of TIMP-1 in the gelatin-unbound fraction together with MMP-3 indicates that the ternary complex was partially dissociated into the MMP-3bulletTIMP-1 complex and free proMMP-9 by weakening of the interaction between proMMP-9 and TIMP-1. This dissociation was more prominent in a higher salt concentration (data not shown). When the proMMP-9bulletTIMP-1 complex was reacted with a one molar excess of MMP-3 for 1 h at 37 °C, all proMMP-9 was converted to an 82-kDa species (80-kDa without reduction) (Fig. 7B). While a larger portion of TIMP-1 was recovered in the gelatin-unbound fraction, about 35% of TIMP-1 was in the bound fraction together with MMP-3. The molecular composition recovered in the latter fraction is therefore the active MMP-9 coupled with an MMP-3bulletTIMP-1 complex. A similar ternary complex and a partial dissociation of proMMP-9 and TIMP-1 were also observed with MMP-1 (Fig. 8). Like in the case of MMP-3, TIMP-1 did not dissociate from proMMP-9 completely even after reaction with one molar excess of MMP-1. Although proMMP-9 remained as a zymogen because MMP-1 is incapable of activating proMMP-9, addition of a catalytic amount of MMP-3 to these samples could fully activate proMMP-9 (data not shown).


Figure 7: Transfer of TIMP-1 from the proMMP-9bulletTIMP-1 complex to active MMPs. The complex (0.1 nmol) was incubated with 45-kDa MMP-3 at 1:0.5, 1:1, and 1:2 molar ratio at 37 °C. After incubation for an indicated period of time, the samples were immediately applied to a gelatin-Sepharose 4B column (1 ml). Unbound (U) or bound (B) fraction were pooled, concentrated with 5% (w/v) trichloroacetic acid, and subjected to Western blotting analyses under reducing conditions (panelB). The first antibodies used were a mixture of sheep anti-(human MMP-9), anti-(human TIMP-1) and anti-(human MMP-3) antibody. PanelA shows the results when the proMMP-9bulletTIMP-1 complex and MMP-3 were applied to the column separately.




Figure 8: Transfer of TIMP-1 from the proMMP-9bulletTIMP-1 complex to MMP-1. The proMMP-9bulletTIMP-1 complex was reacted with MMP-1 at the molar ratio indicated for 2 h. The samples were then analyzed as in Fig. 7except that sheep anti-(human MMP-1) serum was used instead of sheep anti-(human MMP-3) serum.




DISCUSSION

Activation of matrixin zymogens is an important regulatory step in connective tissue matrix turnover. Most proMMPs are activated in vitro by treatment with proteinases, sulfhydryl-reacting agents, mercurial compounds, chaotropic agents, HOCl, low pH, and heat (46, 47, 48, 49) . The recent finding of the progelatinase-TIMP complexes have introduced further complexity in progelatinase activation since TIMP present in the complex can inhibit MMP activities that are required for the final activation step. In this report, we have investigated the mode of activation of the human proMMP-9bulletTIMP-1 complex by APMA and proteinases and the role of TIMP-1 during these processes. The steps involved in the proMMP-9bulletTIMP-1 complex are summarized in Fig. S1.


Figure S1: Scheme 1Steps involved in activation of the proMMP-9bulletTIMP-1 complex by APMA and proteinases. (I), the proMMP-9bulletTIMP-1 binds to and inhibit MMP-3 and other MMPs. The resulting proMMP-9bulletTIMP-1-MMP ternary complex dissociates partially into free proMMP-9 and the TIMP-1-MMP complex. (II), proMMP-9 of the complex is activated by MMP-3 when TIMP-1 is saturated with active MMP. (III), the treatment of the proMMP-9bulletTIMP-1 complex with APMA or trypsin processes proMMP-9 to active forms, but their enzymic activity is inhibited by TIMP-1.



When the proMMP-9bulletTIMP-1 complex was treated with APMA or trypsin, no gelatinolytic activity was detected, although proMMP-9 was converted to lower molecular mass species, which correspond to active forms generated from proMMP-9 free of TIMP-1(35) . The lack of enzymic activity after these treatments was due to the inhibition of the activated MMP-9 by TIMP-1 in the complex (Fig. S1). A low gelatinolytic activity (<6%) detected after the treatment of the complex with APMA is most likely due to the presence of free proMMP-9. Alternatively, it may be due to the contamination of other proMMPs, which bind to TIMP-1 in the complex after APMA treatment, allowing for an equivalent amount of proMMP-9 to be activated. This also emphasizes that the detection of the seemingly active forms of MMP-9 by SDS-PAGE or zymographic analysis alone does not assure that the functionally active enzyme is generated. In this regard, a test for their abilities to interact with alpha(2)M is a useful means to identify proteolytically active enzyme species(31, 35) .

The behavior of the proMMP-2bulletTIMP-2 complex upon APMA treatment is different from that of proMMP-9bulletTIMP-1. In the former case, the disruption of the Cys-Zn interaction by APMA is sufficient for TIMP-2 to bind to the catalytic domain of the activated proMMP-2, and the propeptide of proMMP-2 is not removed(24) . On the other hand, when the proMMP-9bulletTIMP-1 complex is activated by APMA or trypsin, proMMP-9 is processed to low molecular weight active forms, which are then inhibited by TIMP-1. Since the inhibitory N-terminal domain of TIMP-1 in the proMMP-9bulletTIMP-1 complex is free, it is possible that TIMP-1 bound to the C-terminal domain of activated MMP-9 may interact with another activated MMP-9 harboring TIMP-1. Such an interaction may form either a tetrameric complex ((MMP-9bulletTIMP-1)(2)) with 2-fold symmetry or an oligomeric complex ((MMP-9bulletTIMP-1)). However, both the proMMP-9bulletTIMP-1 complex and the activated complex were eluted at the same elution position on the gel permeation chromatography (data not shown), indicating that neither tetrameric nor oligomeric complexes were formed. Nonetheless, our studies do not reveal whether the formation of the active MMP-9bulletTIMP-1 complex is a result of an intra- or intercomplex rearrangement. In the latter case, transient oligomerization should occur, but it is evident that the C-terminal interaction of the proMMP-9bulletTIMP-1 complex becomes weaker when the N-terminal domain of TIMP-1 is occupied by other MMPs (see Fig. 7and 8). This is a contrast to the proMMP-2bulletTIMP-2 complex, which largely remains as a stable ternary complex with another active MMP when it is treated with APMA (24, 50) .

A likely candidate for the in vivo activator of proMMP-9 is MMP-3(21, 31, 32) , but MMP-3 fails to activate the proMMP-9bulletTIMP-1 complex when the amount of MMP-3 is less than a molar stoichiometry (Fig. S1). Under these conditions, MMP-3 is readily inhibited by the complex. Goldberg et al.(21) reported that some of the secreted proMMP-9 forms a covalently bound homodimer or a complex with proMMP-1. These forms of proMMP-9 do not bind to TIMP-1, and they can be readily activated by MMP-3(21) . When the proMMP-9bulletTIMP-1 complex is reacted with a substoichiometric amount of an active MMP, APMA or trypsin was able to generate proteolytically active MMP-9 in proportion to the amount of TIMP-1 bound to an active MMP. On the other hand, the ability of MMP-3 to activate proMMP-9 depends on the balance between TIMP-1 and MMP-3; the saturation of TIMP-1 is prerequisite for MMP-3 to activate proMMP-9 of the complex. The saturation of TIMP-1 may be attained by other active MMPs as shown with MMP-1 in this study.

Once the N-terminal domain of TIMP-1 is coupled with an active MMP, a catalytic amount of MMP-3 can process proMMP-9 to an active 80-kDa MMP-9. Under these conditions, active MMP-9 may be partially bound to the TIMP-1bulletMMP complex ( Fig. 7and Fig. 8and Fig. S1), but it does not give much influence on the enzymic activity of MMP-9. This mode of activation suggests a shift of predominant MMP activity from one type to another. The former MMP activity may be regulated by the TIMP-1 in the proMMP-9bulletTIMP-1 complex, which in turn renders proMMP-9 to be activable. Thus, the expression of specific MMP activities are closely related not only to the temporal gene expression of different types of proMMPs and TIMPs by specific cell types but also to the subsequent activation of proMMPs and the involvement of TIMPs during this process. Such interactions are probably important parameters for determination of precise regulations of extracellular matrix turnover in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR39189 and AR40994. 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.

§
Present address: First Dept. of Surgery, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Japan 830.

To whom 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.

^1
The abbreviations used are: MMP, matrix metalloproteinase; proMMP, precursor of MMP; TIMP, tissue inhibitor of metalloproteinases; alpha(2)M, alpha(2)-macroglobulin; APMA, 4-aminophenylmercuric acetate; DFP, diisopropylphosphorofluoridate; TPA, 12-O-tetradecanoylphorbol-13-acetate; PAGE, polyacrylamide gel electrophoresis; IgG, immunoglobulin G.


ACKNOWLEDGEMENTS

We thank Dr. John L. Fowlkes for critical reading of the manuscript.


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