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The Journal of Immunology, 2002, 168: 2449-2455.
Copyright © 2002 by The American Association of Immunologists

Activation of Macrophage Promatrix Metalloproteinase-9 by Lipopolysaccharide-Associated Proteinases1

Danqing Min*, Anthony G. Moore{dagger}, Michael A. Bain*, Samuel N. Breit{dagger} and J. Guy Lyons2,*

* Kanematsu Laboratories, Sydney Cancer Center, Royal Prince Alfred Hospital, and Faculty of Medicine, University of Sydney, and {dagger} Center For Immunology, St. Vincent’s Hospital and University of New South Wales, Sydney, New South Wales, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LPS induces an up-regulation of promatrix metalloproteinase-9 (proMMP9) gene expression in cells of the monocyte/macrophage lineage. We demonstrate here that LPS preparations are also able to activate proMMP9 made by human macrophages or THP-1 cells via LPS-associated proteinases, which cleave the N-terminal propeptide at a site or sites close to the one cleaved upon activation with organomercurial compounds. LPS-associated proteinases are serine proteinases that are able to cleave denatured collagens (gelatin) and the mammalian serine proteinase inhibitor, {alpha}1-proteinase inhibitor, thereby pushing the balance of extracellular matrix turnover even further toward degradation. A low molecular mass, low affinity inhibitor of MMP9, possibly derived from the propeptide, is generated during proMMP9 activation. However, inhibition of the LPS-associated proteinases had no effect on proMMP9 synthesis, indicating that their proteolytic activity was not required for signaling the up-regulation of the proMMP9 gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The accumulation of macrophages at sites of bacterial infection is an important part of immunity and the inflammatory response. The tissue destruction that accompanies infections of Gram-negative bacteria such as Escherichia coli is facilitated by proteinases, which can originate from the bacteria or from the host inflammatory cells. The matrix metalloproteinases (MMPs)3 are a family of proteinases that can degrade extracellular matrix proteins and are made by macrophages and other cells in response to inflammatory stimuli, such as LPS and cytokines. MMP9, also known as gelatinase B and 92-kDa type IV collagenase, can degrade extracellular matrix components such as collagens and elastins, as well as nonmatrix proteins, including {alpha}1-proteinase inhibitor (1, 2, 3, 4, 5, 6). Neutrophils are another major source of proMMP9 at sites of infection, storing premade enzyme in granules ready to be secreted. Like most MMPs, it is made as a proenzyme that requires the disruption of the interaction of its propeptide with its active site to attain proteolytic activity (7). Activation of proMMPs can be a limiting step in the degradation of extracellular matrix (3). proMMP9 can be activated by serine proteinases such as trypsin and plasmin that cleave the propeptide from the rest of the enzyme (8, 9).

Previous work has identified LPS as a potent inducer of proMMP9 synthesis by macrophages. The mechanism of this induction is not yet clearly understood but, unusually for LPS-induced targets, it does not appear to be dependent on Toll-like receptor 4 (TLR4), because macrophage cell lines derived from the C3H/HeJ strain of mouse, which harbors a null mutation in the TLR4 gene, still up-regulate proMMP9 in response to LPS exposure (10). This raises the question of whether it is LPS itself or some copurifying component that induces proMMP9 gene expression via another pathway. Indeed, LPS-associated proteins have been demonstrated to illicit biological responses from macrophages (11, 12).

In this report, we describe a novel biological activity of LPS preparations: the promotion of extracellular matrix proteolysis by the degradation of {alpha}1-proteinase inhibitor and the activation of proMMP9. This activation of proMMP9 is mediated by LPS-associated proteinases and is distinguished from previously described proteolytic mechanisms by its rapidity and the generation of an MMP9 inhibitor during the activation process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Unless stated otherwise, chemicals were obtained from Sigma Aldrich (St. Louis, MO). The source organism of LPS used in most experiments was E. coli serotype 0111:B4 purified by phenol extraction and ion-exchange chromatography (catalog number L3024). This preparation was verified to have <1% protein content by both Bradford and Lowry methods (13, 14).

Electrophoresis

SDS-PAGE was performed using either Tris-glycine (15) or Tris-tricine (16) buffer systems, as indicated. Silver staining of polyacrylamide gels was done by the method of Merril et al. (17). LPS was fractionated by SDS-PAGE using the Tris-tricine buffer system. LPS and marker proteins were visualized by staining with 0.3 M ZnCl2. Lanes of LPS were cut into 2-mm fractions that were renatured as in zymography (described under Analysis of gelatin-degrading activity). Following renaturation, gel slices were crushed and eluted into MMP assay buffer (50 mM TrisCl, pH 7.5, 10 mM CaCl2, 100 mM NaCl, 0.02% NaN3) or added directly to 20 µl of proMMP9-containing culture medium.

Cell culture

Monocytes were isolated from human peripheral blood by elutriation and cultured as described previously (18). THP-1 human promonocytic leukemia cells were cultured in Ham’s F-12: DMEM (1:1) supplemented with 1 mg/ml BSA, 5 µg/ml of bovine insulin, and 1 µg/ml of human transferrin (19). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air and were subcultured at 1/10 dilution every 3–4 days. In general, experiments were performed 24 h after plating the cells in fresh medium. Cells were seeded at a density of 0.5 x 106/ml/10 cm2. For proMMP9 purification, THP-1 cells were grown to 0.5 x 106/ml and induced by 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), then cultured in 8 liters of serum-free medium in a large culture container with stirring for 2 days. The conditioned medium was then harvested by centrifugation and the cells were resuspended in 8 liters of fresh serum-free medium and cultured without TPA for one more week. After that, the conditioned medium was harvested by centrifugation, pooled with the first harvest, made to 10 µM CGS27023A (a gift from Pharmacia-Upjohn, Milan, Italy), and used for proMMP9 purification on gelatin-Sepharose (Amersham Pharmacia Biotech, Castle Hill, Australia) as described (8). Copurifying tissue inhibitor of metalloproteinase (TIMP)-1 was separated by washing the column-bound proMMP9 with 0.2 M acetic acid (20).

Analysis of gelatin-degrading activity

Gelatin substrate zymography was performed according to the method of Heussen and Dowdle (21) as modified by Lyons et al. (8). Solution-phase assays used heat-denatured rat-tail tendon collagen (22) that had been labeled with 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF; TSI, Tokyo, Japan; Ref. 23). A total of 40 µg of MDPF-labeled gelatin was incubated with the sample at 37°C overnight in MMP assay buffer. EDTA (20 mM) was added to the sample to stop the reaction, and the undigested substrate was precipitated with 0.4 g/ml of ammonium sulfate at 4°C and centrifuged at 14,000 rpm for 30 min. The solubilized products were measured in a Hitachi F-3000 spectrofluorometer (Hitachi, Tokyo, Japan; excitation at 370 nm and emission at 480 nm). The degree of digestion of gelatin was calculated by comparing the value of each sample against the value for the control MDPF-labeled gelatin that was digested completely with 10 µg/ml trypsin. In preliminary experiments, the relationship between the percentage of gelatin solubilized (Y) and the amount of activated MMP9 (X) was found to be parabolic: logY = 0.5logX + 2, for Y <= 50%. This formula was used to convert the raw data into units of activity that were linear with respect to gelatin-degrading activity (1 U = 1 µg of gelatin digested/20 h). All assays were performed such that the percentage of gelatin rendered soluble was <50%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS activates proMMP9 made by macrophages

Macrophages obtained by culturing human peripheral blood monocytes secreted a high level of proMMP9, as detected by gelatin zymography (Fig. 1GoA, lane 1). LPS, although not significantly altering the total amount of proMMP9 produced by macrophages, caused a proportion of it to increase its electrophoretic mobility, consistent with activation of the proenzyme by the removal of the propeptide (Fig. 1GoA, lanes 4–8). This occurred in a dose-dependent manner. The addition of LPS to cell-free macrophage culture supernatants also activated proMMP9, indicating that LPS was acting biochemically on the proMMP9 in the culture medium, rather than inducing the macrophages to produce an activating substance (Fig. 1GoB). Moreover, the addition of LPS to purified, TIMP-1-free proMMP9 that had been isolated from the culture medium of TPA-differentiated THP-1 cells also activated the proMMP9 (Fig. 2GoA), indicating that the LPS acted directly on the proMMP9 and not via an intermediary target, such as by activating another proteinase that in turn cleaved proMMP9.



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  		FIGURE 1. LPS induces synthesis of proMMP9 by macrophages and activates it. Human macrophage conditioned media were analyzed for MMP9 by gelatin substrate zymography. A, Macrophages were cultured for 24 h in medium containing LPS at the indicated concentrations. Densitometry was used to quantitate the MMP9 species present in the LPS-treated cultures. The band intensities of proMMP9 (black) and active MMP9 (gray) are expressed as percentages of the proMMP9 band in lane 1 and plotted in the bar graph beneath the respective samples. B, Conditioned media from control macrophage cultures were incubated for 1 h at 37°C with the indicated concentration of LPS.

 


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  		FIGURE 2. Activation of proMMP9 by LPS is rapid. Gelatin substrate zymograms are shown of purified proMMP9 that had undergone activation with 50 µg/ml LPS (A), 1 mM APMA (B), or 5 µg/ml trypsin (C) at 37°C (lanes 1–5) or 21°C (lanes 6–11) for the time indicated above each lane. Lane P, The purified proMMP9 before activation. Lane M, THP-1-conditioned medium that had been completely activated with the agent by incubation for 24 h at 37°C.

 
The activation of proMMP9 by LPS occurred quickly, and most of the activated MMP9 was produced within 5 min at either 37 or 21°C (Fig. 2GoA). The degree of activation was dependent on the concentration of LPS used (Fig. 1Go, A and B). Unlike activation by the organomercurial compound, p-aminophenylmercuric acetate (APMA; Fig. 2GoB), or by trypsin (Fig. 2GoC), active MMP9 produced by the action of LPS did not accumulate progressively during the hour following the addition of the activating substance (Fig. 2GoA). Much longer incubations with LPS demonstrated a very slow accumulation of active MMP9 over 24 h (data not shown). This slow, second phase of activation may not have been due to LPS itself, but rather to the cleavage of proMMP9 by the proportion of MMP9 that had been rapidly activated. Thus, activation of proMMP9 by LPS was concentration-dependent, very rapid and incomplete.

The LPS-activated MMP9 had an electrophoretic mobility very similar to that of MMP9 activated by organomercurials such as APMA (Fig. 2Go), which causes the removal of 9 kDa of the N-terminal propeptide to give to an 83-kDa species (8, 24, 25). To determine whether the LPS-induced cleavage occurred at the N terminus or the C terminus of proMMP9, medium containing proMMP9 was treated sequentially with LPS and APMA. If the LPS-induced removal of 9 kDa had occurred at the C terminus of the protein, then activation with APMA would cause the removal of an additional 9 kDa, resulting in the appearance of a 74-kDa species (Fig. 3Go, A and B). Removal of 9 kDa from either end would retain proteolytic activity in gelatin zymograms, because the cleavage would occur outside the catalytic domain (3). Activation with APMA either before or after LPS activation produced an 83-kDa species, but no 74-kDa species, demonstrating that the LPS-mediated cleavage removed the N terminus, and not the C terminus or glycosides from the protein (Fig. 3GoC).



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  		FIGURE 3. LPS cleaves the N terminus of proMMP9. A, Schematic representation of intact Mr 92-kDa (k) proMMP9 with N and C termini indicated. B, Schematic representations of the proMMP9 fragments and their sizes predicted by activation with LPS and APMA. Fragments that would produce proteolytic bands in the zymogram are filled in black. C, Gelatin substrate zymogram of proMMP9-containing culture medium either not activated (lane 5) or activated with APMA followed by LPS (lane 1), with LPS followed by APMA (lane 2), with LPS (lane 3), or with APMA (lane 4). Samples of LPS only (lane 6) and APMA only (lane 7) were analyzed as controls.

 
proMMP9 propeptide cleavage by LPS is mediated by a high molecular mass serine proteinase

MMP proenzymes can be activated by processes that interfere with the interaction of the "switch" Cys of the propeptide with the active site zinc (7), including oxidation of the Cys sulfhydryl, disruption of the tertiary structure by chaotropes or detergents and proteolysis of the propeptide. The detergent-like dual lipid-carbohydrate nature of LPS or the presence of an oxidizing component in it could conceivably cause activation of proMMP9, which would then cleave off its own propeptide. However, the addition of the MMP inhibitor, CGS27023A, up to 50 µM did not inhibit LPS-mediated activation of proMMP9 (Fig. 4GoA), indicating that autolysis by MMP9, which is required for propeptide cleavage induced by oxidants and detergents, is not involved in the activation process. To determine whether the LPS contained a proteolytic activity that was required for proMMP9 propeptide cleavage, protease inhibitors were screened for their ability to inhibit the reaction (Fig. 4GoB, lanes 2–12). 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and PMSF (1 mM) were inhibitory, whereas EDTA and N-ethyl maleimide (NEM), inhibitors of metallo- and cysteine proteinases, respectively, were not inhibitory, consistent with a serine proteinase in LPS being responsible for cleaving the proMMP9 propeptide. N-Tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone (TLCK) were poor inhibitors of proMMP9 activation.



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  		FIGURE 4. Effect of inhibitors on LPS-associated proteases. LPS was preincubated with protease inhibitors at 37°C for 1 h and/or boiled. It was then assessed when present at 50 µg/ml for its ability to activate proMMP9 in culture medium, indicated by a shift in electrophoretic mobility in gelatin substrate zymograms (A–C), or to cleave {alpha}1-proteinase inhibitor (D). A, MMP inhibitor CGS27023A (CGS) was present during activation at 10–50 µM, as indicated above each lane. B, EDTA and EGTA were used at 5 and 10 mM, respectively, and AEBSF and PMSF were used at 0.1 mM in lanes 7 and 10, respectively. In all other lanes, the inhibitors were used at 1 mM. Phen (lane 12) is 1,10-phenanthroline. The formation of the pro- and active MMP9 dimer species (their positions indicated at right) is inhibited by NEM, suggesting that they form after the addition of zymogram sample buffer. C, Conditions were the same in those in B, except that the LPS was boiled for 10 min before addition of inhibitors and proMMP9 activation. D, LPS was incubated with 5 µg of {alpha}1-proteinase inhibitor overnight and analyzed by SDS-PAGE and stained with Coomassie brilliant blue. P + T + A, 0.1 mM each of PMSF, TLCK, and AEBSF. Otherwise, treatments were the same as in B and C.

 
Gelatin zymography was used to examine LPS for AEBSF-sensitive proteinases that may have been responsible for the proMMP9 propeptide cleavage (Fig. 5GoA). A strong, discrete band of proteolysis corresponding to a molecular mass of 22 kDa was evident, as was a band of activity of >200 kDa that sometimes resolved into two broad bands (lane 1). A discrete band at 180 kDa was also present in some zymograms, but not other zymograms of the same sample. The 22-kDa band migrated more slowly in Tris-tricine gels, comigrating with the 29-kDa molecular mass marker (Fig. 5GoB). Preincubation of the LPS with AEBSF abolished the activity of all of the proteases, whereas preincubation with NEM did not (Fig. 5GoA, lanes 3 and 4), indicating that all of the zymogram bands represented serine proteinases. TLCK inhibited the 22- and 180-kDa bands, whereas EDTA eliminated the activity of the 180-kDa band, but had no significant effect on the other bands. N-tosyl-L-phenylalanine chloromethyl ketone had no effect on any of the proteinases. Boiling the LPS for 10 min, which destroys most of the proMMP9-activating activity (Fig. 4GoC), abolished all but the 22-kDa proteolytic band, which retained its original intensity (Fig. 5GoA, compare lanes 1 and 7). However, the inhibitor profile of the boiled sample suggested that the 22-kDa band was partially inhibited by boiling and that the zymography process had renatured it, because a substantial proportion of its activity was resistant to AEBSF and TLCK (Fig. 5GoA, lanes 10 and 12), which require an active enzyme to bind covalently to serine proteinases at their active sites. The proteinase responsible for proMMP9 activation was sensitive to boiling and AEBSF, but insensitive to TLCK and EDTA (Fig. 4Go, B and C), properties identical with the >200-kDa band observed by gelatin zymography (Fig. 5GoA).



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  		FIGURE 5. SDS-PAGE analysis of LPS-associated proteinases. A, LPS was treated with protease inhibitors as described in Fig. 4Go and analyzed for proteases by gelatin substrate zymography. The samples in lanes 7–12 were boiled for 10 min before treatment with protease inhibitors. B, LPS (150 µg) was separated by SDS-PAGE in a Tris-tricine buffer system in a 5% gel and stained with 0.3 M ZnCl2 to visualize the LPS (middle panel). It was cut into 2-mm fractions that were renatured, crushed, and mixed with 20 µl of TPA-stimulated THP-1-conditioned medium which were then analyzed by gelatin substrate zymography. The MMP9-containing part of the zymograms are shown in the right panel beside the corresponding 2-mm fraction of the LPS gel; the migration in the zymogram was from left to right. The positions of molecular mass markers in the LPS gel are indicated at the far right. A gelatin zymogram of LPS using the Tris-tricine buffer system is shown in the left panel. C, Various amounts of LPS (indicated at the top of each lane) were separated by SDS-PAGE in a Tris-tricine buffer system in a 7.5% gel and silver stained. The positions of molecular mass markers are indicated at right.

 
As well as activating proMMP9 and degrading gelatin, LPS-associated proteinases were able to degrade {alpha}1-proteinase inhibitor (Fig. 4GoD), a plasma protein that inhibits plasmin and other serine proteinases. The cleavage pattern was similar to that obtained when active MMP9 digested {alpha}1-proteinase inhibitor, which causes inactivation of the latter (8, 26). The inhibitor profile of {alpha}1-proteinase inhibitor degradation was similar to that for proMMP9 activation, except that it was resistant to 1 mM PMSF (Fig. 4GoD, lane 7), suggesting that a different proteinase was responsible.

LPS was fractionated by SDS-PAGE using the Tris-tricine SDS-PAGE buffer system to determine the molecular mass of the proteinase(s) responsible for proMMP9 activation. When 150 µg of LPS was loaded per lane, a strong peak of activation was consistently seen near the top of the polyacrylamide gel, indicating that the activating substance had a molecular mass well in excess of 200 kDa (the third fraction from the top in Fig. 5GoB). A second peak of activity was usually seen just below the 200-kDa marker. The use of the Tris-tricine electrode buffer effectively separated these high molecular mass peaks of activity from the bulk of the LPS (Fig. 5GoB), whereas Tris-glycine produced a smear of LPS that reached the top of the gel (data not shown). The relative mobilities of the gelatinolytic proteinases seen in zymograms also altered in Tris-tricine. This enabled the resolution of the proMMP9-activating activity from the main high molecular mass gelatinolytic band seen in zymograms, the former peaking in fraction 3, whereas the latter was confined to fractions 1 and 2 (Fig. 5GoB). Thus, the proMMP9-activating proteinase was not a major source of the gelatin-degrading activity in LPS preparations.

To determine how potent was the main peak of proMMP9 activating material, the LPS was analyzed for its protein content by protein assays and electrophoresis. The amount of protein in the original LPS preparation was found by Bradford and Lowry methods to be ~1% by weight, in agreement with the supplier’s specifications. The amount of protein in the part of the SDS-PAGE gel containing the proMMP9-activating activity was very small, since none was detected by staining a lane loaded with 100 µg of LPS with Coomassie brilliant blue G250, which has a sensitivity of 0.3–1 µg per band (27) or with ZnCl2 (Fig. 5GoB), which has a sensitivity of 10–100 ng per band. Silver staining of the LPS preparations showed the characteristic ladder representing the varying numbers of sugar subunits added to the lipid A core (Fig. 5GoC). A smear of silver-stained material (possibly protein) was visible in the region mainly responsible for proMMP9 activation at high loadings of LPS (>=25 µg; Fig. 5GoC, lanes 1–3), but not at lower loadings (>=12.5 µg; Fig. 5GoC, lanes 4 and 5). Given that silver staining of proteins in polyacrylamide gels is sensitive to 2–5 ng protein (27), the activating substance represented <0.04% by mass of the LPS. Thus, <30 ng or 1.5 x 10-13 mol of total protein was required to activate the proMMP9 in 20 µl culture medium (containing ~2 µg or 2 x 10-11 mol of proMMP9) to the degree seen in Fig. 5GoB.

MMP9 activated by LPS-associated proteinase has weak gelatin-degrading activity due to the generation of an inhibitor

To confirm that the removal of the propeptide by LPS resulted in active MMP9, gelatin-degrading activity was assayed by a solution-phase fluorescent assay (Fig. 6Go). To prevent the gelatin-degrading activity intrinsic to the LPS from interfering with the MMP9 activity, TLCK (0.1 mM) and PMSF (0.1 mM) were added after activation, but before the addition of the gelatin substrate. This combination of serine proteinase inhibitors completely inhibited degradation of gelatin by LPS when present during the assay (Fig. 6GoA, bar 5). Purified, TIMP-1-free proMMP9 had some proteolytic activity (bar 1), presumably due to the proportion of the proenzyme molecules having their switch Cys in the "open" position in the equilibrium state (28). Treatment with APMA or trypsin led to a 3- to 7-fold proteolytic activation (bars 3 and 4) and was accompanied by progressively increasing propeptide cleavage (Fig. 2Go), in agreement with other studies (8, 9, 24). In contrast, propeptide cleavage by LPS was accompanied by a very low level of proteolytic activation (<2-fold; Fig. 6GoA, bar 2), occurred extremely rapidly, and did not increase over time (Fig. 2Go).



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  		FIGURE 6. Gelatinolytic activity of LPS-activated proMMP9. A, proMMP9 (250 ng) was activated by exposure to LPS, APMA, or trypsin (lanes 2–4, respectively), and the proteolytic activity generated was measured against a fluorescent gelatin substrate. Unactivated proMMP9 (lane 1) and activators without added proMMP9 (lanes 5–7) were included as controls. B, proMMP9 (20 ng) was activated with LPS, added to serine proteinase inhibitors, and fractionated over a 20 kDa molecular mass cut-off filter. The gelatinolytic activity of the retentate (lane 1), filtrate (lane 2), combined retentate and filtrate (lane 3), and sample before filtration (lane 4) were measured against a fluorescent gelatin substrate. proMMP9 only (lanes 5–8) and LPS only (lanes 9–12) were treated similarly. In other assays, the retentate from the LPS-activated proMMP9 sample was added to the filtrate from the proMMP9-only sample (lane 13) or the filtrate from the LPS-only (lane 14) sample.

 
The limited extent of MMP9 proteolytic activity accompanying LPS activation was not caused by an inhibitor present in the LPS itself, because the addition of LPS to MMP9 activated by APMA or trypsin or MMP9 that had activated spontaneously during purification was not inhibitory (data not shown). However, when LPS-activated MMP9 was fractionated by ultrafiltration over a molecular mass cut-off 20-kDa membrane, the retentate, which contained the active MMP9, had enhanced proteolytic activity compared with the unfractionated material (Fig. 6GoB, compare bars 1 and 4) similar to that of trypsin-activated enzyme. The filtrate was able to inhibit this enhanced gelatinolytic activity (bar 3), suggesting that a low molecular mass inhibitor of MMP9 was generated during the propeptide cleavage process. Filtrates from control ultrafiltration experiments, in which LPS and proMMP9 were fractionated separately, did not contain inhibitory activity (bars 13 and 14), demonstrating that the <20-kDa inhibitor was generated during activation of proMMP9, and was not present in either the LPS or the proMMP9 starting material.

LPS-associated proteolytic activity is not required for induction of proMMP9 gene expression

The finding by Zhang et al. (29) that an endogenous serine proteinase inhibitor was able to suppress proMMP9 production by macrophages prompted us to hypothesize that the serine proteinases in LPS were partly responsible for inducing proMMP9 gene expression by LPS. However, pretreatment of LPS preparations with AEBSF, PMSF, and TLCK had no effect on proMMP9 induction in THP-1 cells when the LPS was used at 50 µg/ml (data not shown). Experiments in which the protease inhibitor-pretreated LPS was used at 5 µg/ml also showed no difference from control LPS. Thus, the LPS-associated proteinases did not induce proMMP9 production, at least via a mechanism that required proteolysis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, E. coli LPS preparations have been demonstrated to promote extracellular matrix degradation by multiple routes: by activating the proMMP9 produced, by degrading denatured collagen (gelatin), and by degrading {alpha}1-proteinase inhibitor. This massive shift in balance from homeostasis toward extracellular matrix breakdown could be important in the pathological tissue destruction that accompanies infections by E. coli and other Gram-negative bacteria in tissues such as those of the genitourinary tract and gingivae. Active MMP9 would be generated and {alpha}1-proteinase inhibitor would be depleted at the bacterial cell surface and areas surrounding infections where high concentrations of LPS and its high molecular mass-associated proteinases accumulate.

The present work demonstrates that LPS preparations contain associated proteinases that can have a biological effect: namely, the activation of proMMP9 and degradation of {alpha}1-proteinase inhibitor and gelatin. The proteolytic activities of these LPS-associated proteinases were apparently not directly involved in inducing expression of the proMMP9 gene, as determined by the resistance of this effect of LPS to proteinase inhibitors. Nevertheless, some other biological effects of LPS preparations are apparently not mediated by the binding of lipid A to TLR4, but rather by the action of other components on Toll-like receptor 2 or other signal transducers (11, 12, 30, 31, 32), and may be mediated in a nonproteolytic way by the LPS-associated proteinases or by other components.

The activation of proMMP9 by LPS-associated proteinases has not been reported previously. The relatively high concentration of LPS required to see this effect suggests that it would occur in vivo only where high concentrations of LPS exist, such as at the bacterial cell surface and in tissues adjacent to gross infection. It remains a possibility, however, that the high molecular mass proteinases responsible for activating proMMP9 may be in a greater abundance in vivo than in LPS preparations, which may lose or inactivate part of their activity during the harsh purification procedures used to isolate them, including hot phenol extraction. The identities of the multiple proteinases detected in LPS preparations are not yet known. LPS is a heterogeneous complex of lipid A covalently bound to two glycosidic domains of variable composition (33) and, at least in Porphyromonas gingivalis, proteinases can be covalently bound to LPS glycosides (34), which could account for the copurification and high molecular mass of the proMMP9-activating proteinase. The proteinase responsible for activating proMMP9 is apparently not one of those detected by gelatin zymography, as SDS-PAGE could separate them (Fig. 5Go). It also appears to be distinct from the proteinase that degrades{alpha}1-proteinase inhibitor, as they display distinct susceptibilities to PMSF (Fig. 4Go).

Several features of the LPS activation of proMMP9 are unusual for proMMP activators. The cleavage of the N-terminal propeptide by the >200 kDa LPS-associated proteinase of E. coli gives rise to an active MMP9 species with low proteolytic activity, similar to that obtained by activation with the gingipains of P. gingivalis, which cleave the proMMP9 propeptide at the Lys73-Ala74 peptide bond (35). We have shown here that this low activity is not intrinsic to the activated MMP9, but is caused by the generation of a low molecular mass inhibitor. By analogy with MMP2 propeptide sequences (36), it is possible that the propeptide of proMMP9 retains some inhibitory activity following its cleavage from the active enzyme, and that this is the low-affinity inhibitor being generated during activation with LPS and separated from the active enzyme by ultrafiltration. Inactivation of this inhibitor by another proteinase or simple diffusion from the site of proMMP9 activation in vivo would result in completely active enzyme. The speed and incompleteness of the proMMP9 cleavage by LPS activation resembled that of a titratable, rather than catalytic activator, and distinguished LPS activation from activation with APMA or trypsin, which continued to activate over time. Possibly, the LPS-associated proteinase was somehow inactivated rapidly following proMMP9 activation. Repeated attempts to obtain N-terminal sequence of the LPS-activated MMP9 were not successful, suggesting either that two or more nearby sites were cleaved, or that the N terminus was blocked during activation. While this work was being undertaken, a report was published that LPS could cleave proMMP2 (37). Unfortunately, it is not clear from that work where in the proMMP2 protein this cleavage took place or whether the gelatinolytic activity observed was due to active MMP2 or to the LPS-associated proteinases.

{alpha}1-Proteinase inhibitor inhibits the activity of proMMP9-activating serine proteinases such as trypsin and plasmin (38), and so its degradation by the LPS-associated proteinases would indirectly facilitate proMMP9 activation through serine proteinases. Serine proteinases themselves are able to degrade certain components of the extracellular matrix, a process that would be enhanced by the degradation of {alpha}1-proteinase inhibitor by LPS-associated proteinases and activated MMP9. Additionally, it has recently been demonstrated that {alpha}1-proteinase inhibitor fragments can up-regulate proMMP9 and TNF-{alpha} production (39), as well as attracting neutrophils, another source of proMMP9, providing yet another procatabolic mechanism. Thus, LPS preparations provide a biochemical as well as a cell-signaling mechanism for enhancing extracellular matrix destruction.


   Footnotes
 
1 This work was supported in part by a grant from the National Health and Medical Research Council (to J.G.L.) and New South Wales Health Research and Development Infrastructure Grants to the Kanematsu Laboratories and the Center forImmunology. Back

2 Address correspondence and reprint requests to Dr. J. Guy Lyons, Kanematsu Laboratories, Sydney Cancer Center, Royal Prince Alfred Hospital, Missenden Road, Camperdown, New South Wales 2050, Australia. E-mail address: guy{at}kan.rpa.cs.nsw.gov.au Back

3 Abbreviations used in this paper: MMP, matrix metalloproteinase; proMMP9, promatrix metalloproteinase-9; TLR4, Toll-like receptor 4; TPA, 12-O-tetradecanoylphorbol 13-acetate; MDPF, 2-methoxy-2,4-diphenyl-3(2H)-furanone; APMA, p-aminophenylmercuric acetate; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; NEM, N-ethyl maleimide; TLCK, N-tosyl-L-lysine chloromethyl ketone; TIMP, tissue inhibitor of metalloproteinase. Back

Received for publication August 13, 2001. Accepted for publication December 21, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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