Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0203086 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:88-94.)
© 2003 by Society for Leukocyte Biology

Proteinase-3 directly activates MMP-2 and degrades gelatin and Matrigel; differential inhibition by (-)epigallocatechin-3-gallate

Elga Pezzato*, Massimo Donà*, Luigi Sartor*, Isabella Dell’Aica*, Roberto Benelli{dagger}, Adriana Albini{dagger} and Spiridione Garbisa*

* Department of Experimental Biomedical Sciences, Medical School, Padova, Italy; and
{dagger} Molecular Biology Laboratory, National Institute for Research on Cancer, IST, Genova, Italy

Correspondence: Spiridione Garbisa, Department of Experimental Biomedical Sciences, Medical School, Viale G. Colombo 3, 35121 Padova, Italy. E-mail: garbisa{at}unipd.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase-3 (PR-3), a serine-proteinase mainly expressed by polymorphonuclear leukocytes (PMNs), can degrade a variety of extracellular matrix proteins and may contribute to a number of inflammation-triggered diseases. Here, we show that in addition to MatrigelTM components, PR-3 is also able to degrade denatured collagen and directly activate secreted but not membrane-bound pro-MMP-2, a matrix metallo-proteinase instrumental to cellular invasion. In contrast, following addition of purified PR-3 or PMNs to HT1080 tumor cells, dose-dependent inhibition of in vitro MatrigelTM invasion is registered. (-)Epigallocatechin-3-gallate (EGCG), the main flavanol in green tea and known to inhibit inflammation and tumor invasion, exerts dose-dependent inhibition of degradation of gelatin (IC50<20 µM) and casein, which is directly triggered by PR-3. The presence of EGCG does not modify the colocalization of MMP-2 and exogenous PR-3 at the cell surface and does not restrain secreted pro-MMP-2 and pro-MMP-9 activation or degradation of a specific, synthetic peptide by PR-3. These results add new activities to the list of those exerted by PR-3 and indicate a differential inhibition as a result of EGCG.

Key Words: neutrophils • gelatinolysis • EGCG


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase-3 (PR-3) is a 29-kDa neutral serine-protease expressed by some human tissues, such as lung, heart, and brain [1 ], and by immunocompetent cells [2 , 3 ]. The azurophil granules of human polymorphonuclear leukocytes (PMNs) represent its most abundant source, in which it is stored together with neutrophil elastase, cathepsin G (Cat.G), and enzymatically inactive azurocidin [4 , 5 ]. PR-3, also known as myeloblastin, can degrade a variety of extracellular matrix (ECM) proteins, such as elastin, fibronectin, laminin, vitronectin, and type IV collagen [6 7 8 ], and show no or minimal activity against the interstitial types I and III collagens [5 ].

Physiologically, the proteolytic activity of PR-3 may facilitate the extravasation of neutrophils from the bloodstream at sites of inflammation [9 ] or assist in the digestion of phagocytized microorganisms [10 ]. PR-3 can activate gelatinases A and B [matrix metallo-proteinase (MMP)-2 and MMP-9], which degrade basement membrane barriers and are instrumental in inflammation, angiogenesis, cancer invasion, and metastasis [11 ]. Although MMP-9 can be directly activated by PR-3 [12 ], it has been claimed that activation of MMP-2 requires the concerted action of PR-3 and the membrane-anchored metallo-proteinase membrane type 1 (MT1)-MMP [13 ].

Pathologically, PR-3 is involved in a number of diseases characterized by disruption of normal tissue architecture, most notably, cystic fibrosis [14 ], Wagener’s granulomatosis [15 ], vasculitis and rheumatoid arthritis [16 ], glomerulonephritis [17 ], and adult respiratory distress syndrome [18 ]. Following intratracheal instillation of PR-3, emphysematous lesions readily develop in the lungs of hamster [19 ], suggesting that the elastolytic property of PR-3 may be instrumental in the development of pulmonary emphysema. PR-3 is also overexpressed in a variety of acute and chronic myeloid leukemia cells [20 , 21 ].

This enzyme is physiologically counterbalanced by endogenous, serine-protease inhibitors {alpha}1-proteinase inhibitor ({alpha}1-PI), {alpha}2-macroglobulin, the 6-kDa inhibitory domain of elafin, and monocyte/neutrophil elastase inhibitor [5 , 22 ] but any enzyme/inhibitor imbalance may lead directly to increased lysis of ECM molecules and risk of tissue injury. In particular, {alpha}1-PI deficiency is the most prevalent, potentially fatal hereditary disease in white individuals and an important risk factor for pulmonary emphysema [23 ]. Regarding treatment, in many cases, exogenous inhibitors would be first-choice drugs, and direct {alpha}1-PI replacement is one potential therapeutic approach currently under investigation.

Some vegetable, secondary metabolites have recently been shown to exert good inhibitory activity against metallo- and serine-proteases. In particular, the most abundant catechin of green tea, (-)epigallocatechin-3-gallate (EGCG), inhibits, at micromolar concentrations and in a dose-dependent manner, the activity of gelatinases A and B [24 ], MT1-MMP [25 ], and leukocyte elastase (LE) [26 ] and restrains inflammation [27 ] and tumor invasion [28 ].

Given the growing role of PR-3 in numerous and severe pathologies, we investigated the possible contribution of PR-3 to ECM degradation and cell invasion and the possible inhibitory effect of EGCG on PR-3 activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
PR-3 and rabbit anti-human PR-3 antibody were from Elastin Products Co. (Owensville, MO); mouse anti-human MMP-2 antibody, from Oncogene (Cambridge, MA); MeO-Suc-K(Pic)APV-pNA, from Bachem AG (Bubendorf, Switzerland), MatrigelTM, from Collaborative Research, Beckton Dickinson (Bedford, MA); and polycarbonate filters, from Millipore (Bedford, MA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). EGCG was purified ≥95%.

Cells
HT1080 human fibrosarcoma cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany). The medium was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Labtek Eurobio, Corsio, Italy), and the cells were incubated in 5% CO2 in air at 37°C.

PMNs were isolated under endotoxin-free conditions from buffy coats of healthy donors, following a single-step separation procedure [23 ], as previously described in detail [24 ]. The resulting cell population contained 96–98% neutrophils with traces of eosinophils and mononuclear cells. The cells were suspended in serum-free DMEM and activated by gentle shaking for 60 min.

Cell membrane extraction
After rinsing with phosphate-buffered saline (PBS), the cells were harvested with 2 mM EDTA, centrifuged 5 min at 500 g, and rinsed twice with PBS. The pellet was resuspended in 200 µl 0.1% digitonin (Sigma Chemical Co.), 0.5 M NaCl, 20 mM Hepes, pH 7, and incubated 10 min at 4°C. Following 5 min centrifugation at 1200 g, the pellet was rinsed three times with the above buffer, resuspended in 1% digitonin, 0.15 M NaCl, and 20 mM Hepes, pH 7, and incubated 2 h at 4°C with gentle motion. The sample was then centrifuged 5 min at 1200 g, and the cell membrane, protein-containing supernatant was collected and lyophilized.

Zymographic analysis
Without heating, samples underwent electrophoresis in 0.1% gelatin-containing polyacrylamide (% specified in figure legends) gels in the presence of sodium dodecyl sulfate (SDS) under nonreducing conditions, as previously described [24 ]. After electrophoresis, the gels were washed twice for 15 min with 2.5% Triton X-100 and incubated overnight at 37°C in Tris buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.4). For gelatinolytic activity inhibition assays, compounds were freshly solubilized in ethanol and diluted in Tris buffer used for developing the zymogram. For inhibition assays, the gel slab was then cut into slices corresponding to the lanes and put in different tanks containing the stated concentrations of inhibitors.

The gels were then stained for 30 min with 30% methanol/10% acetic acid containing 0.5% Coomassie brilliant blue R-250 and were destained in the same solution without dye. Clear bands on the blue background represent areas of gelatinolysis. Digestion bands were quantitated using an image analyzer system with GelDoc 2000 and Quantity One software (Bio-Rad, Hercules, CA).

Synthetic substrate and casein degradation by PR-3
PR-3 was also assayed using the chromogenic substrates MeO-Suc-K(Pic)APV-pNA and MeO-Suc-AAPV-pNA or casein–fluorescein isothiocyanate (FITC) [29 ]. Stock solutions were prepared as follows: PR-3 (0.1 mg/ml) in 0.1 M glycine, 0.1 M NaCl, pH 3.2 (buffer 1); EGCG (10x the final concentration) in 0.1 M Hepes, 0.5 M NaCl, and 10% dimethylsulfoxide (DMSO), pH 7.5 (buffer 2); chromogenic substrates (20 mM) in DMSO; casein–FITC (0.5 mg/ml) in 50 mM Tris-HCl and 1 M NaCl, pH 7.8.

PR-3 solution (20 µl) was mixed with 10 µl EGCG solution, and the final volume adjusted to 80 µl with 50 mM Na+/K+ phosphate buffer containing 0.05% Triton-X, 0.5% DMSO, and 0.125 M NaCl, pH 7 (buffer 3). This mixture was preincubated 30 min at 4°C, and then 20 µl substrate diluted in buffer 3 was added (final concentration, 200 µM). The incubation was continued at 37°C in flat-bottom, 96-well, tissue-culture plates; the colorimetric reaction was quantitated at 405 nm using a Titertek Multiskan PLUS (Flow Laboratories) at 30-min intervals up to 3.5 h, and the fluorometric reaction was quantitated using a Fluoro Count microplate reader (Packard, Toronto, Canada) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The results were expressed as mean ± SD of triplicate reaction (appropriate controls subtracted).

MatrigelTM degradation by PR-3 or PMNs
The degradative activity of purified PR-3 or PMNs on basement membrane components was analyzed using gelified MatrigelTM solution, which was freshly prepared in distilled water at a concentration of 0.84 mg/ml, and 50 µl was layered on polyvinylpyrrolidone-free polycarbonate filters (8 µm pore size; Millipore), precoated with 0.1% gelatin (as in Boyden-chamber assay, see below). Gelification was obtained at room temperature (RT) under air flow and in petri dishes for better solvent evaporation, and the filters were then transferred into 15-mm wells. EGCG and PR-3 solutions or EGCG and 1.2 x 105PMN were then added, and the volume was adjusted to 140 µl as above; following 30 min preincubation at 4°C, 60 µl buffer 3 was added, and the incubation continued at 37°C under gentle shaking for 3 or 5 h, respectively. The supernatant and the MatrigelTM solubilized with 100 µl electrophoresis sample buffer were lyophilized and reconstituted in distilled water and analyzed by standard SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.

Gelatinase activation
To study the effect of PR-3 on activation of membrane-bound and secreted gelatinases in cell culture, HT1080 cells at 70% confluence were rinsed three times with PBS and incubated at 37°C with (2.5 µg/ml) or without PR-3 in serum-free medium. After 2 h, the media were collected and clarified, the cell membranes extracted as described above, and aliquots analyzed by gelatin zymography.

To study the activity of PR-3 on pro-gelatinases MMP-2 and MMP-9, pro-gelatinase-containing culture media were used [24 , 30 ]. PR-3 was diluted in 0.1 M Hepes, 0.1 M NaCl, 10 mM CaCl2, 0.005% Triton X-100, and 5% DMSO, pH 7.5 (buffer 4), and added to the media at increasing concentrations (0–50 µg/ml) in the presence of 5 mM N-ethyl-maleimide (NEM); the sample was incubated 5 h at 37°C. After mixing with electrophoresis sample buffer 4x, aliquots of the samples were analyzed by gelatin zymography. The same procedure was used to assay LE and Cat.G (50 mU/ml).

PMN-triggered activation of gelatinase was also studied as follows: Activated PMNs were coincubated with adherent HT1080 cells under gentle motion for 2 and 4 h at 37°C in 1:1 and 10:1 ratio and were removed by mechanical shock and rinsing (efficacy, >95%, as judged by microscopy). The same experiment was carried out substituting activated PMNs with their clarified, conditioned medium. Conditioned media and membrane extracts were analyzed by gelatin zymography.

To study the effect of EGCG on PR-3-driven MMP-2 and MMP-9 activation, 50 µg/ml PR-3 in buffer 1 was premixed 30 min at 4°C with 5 and 15 µM EGCG in buffer 4. The mixture (25 µl final volume) was then added to a 15 µl of gelatinase-containing medium, and the pH was verified as neutral; after 5 h incubation at 37°C in the presence of 5 mM NEM, the samples were directly examined by gelatin zymography.

Confocal immunofluorescence microscopy
Cells were seeded on glass coverslips and incubated at 4°C with EGCG (1 and 10 µM) and PR-3 (30 µg/ml) in PBS with Ca2+, Mg2+, and 1% bovine serum albumin (BSA) in the following three combinations: T0, EGCG (for 60 min) - T1, PR-3 (for 30 min); T0, PR-3 (for 30 min) - T1, EGCG (for 60 min); and T0, EGCG and PR-3 (for 90 min). The cells were then fixed with 4% paraformaldehyde for 30 min. After washing with PBS and blocking with 0.3% BSA in PBS, the cells were incubated overnight at 4°C with a mix of two first antibodies (rabbit anti-human PR-3 and mouse anti-human MMP-2 antibodies, 1:8000 and 1:100, respectively), washed four times with PBS, incubated with the appropriate secondary antibodies [Cy3-conjugate sheep anti-rabbit immunoglobulin G (IgG), 1:1000, and FITC-conjugate goat anti-mouse IgG, 1:100] for 1 h at RT, and washed four times with PBS. Coverslips were then mounted on microscope slides over a drop of fluorescent-mounting medium (Dako, Copenhagen, Denmark). Staining was visualized using a Bio-Rad confocal microscope.

Modified Boyden-chamber assay
The invasive behavior of HT1080 human fibrosarcoma cells in the presence of exogenous PR-3 and EGCG was tested using the modified Boyden-chamber assay. MatrigelTM and gelatin were used as the matrix for cells to migrate through toward a chemoattractant represented by culture medium conditioned by NIH-3T3 cells [24 ]. Polyvinylpyrrolidone-free polycarbonate filters (8 µm pore size) were precoated by immersion in gelatin solution (0.1%) and coated with MatrigelTM (50 µl 0.66 mg/ml; concentration lower than in MatrigelTM degradation assay to allow invasion). Following seeding of 1.2 x 105 cells onto the filters and 4 h incubation in serum-free medium without and with PR-3 (0.6 and 6 µg/ml), nonmigrated cells on the upper surface of the filter were removed. The filter was rinsed in water and fixed with 100% ethanol for 30 s, stained with toluidine blue for 10 min, and scanned at 600 pixels per inch. The cells that actively migrated to the under-surface of the filter were quantitated using NIH-Image 1.61 software, and the results of triplicate experiments were averaged after background subtraction. Control experiments were performed in the absence of chemoattractant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Degradation of gelatin, casein, and synthetic substrate by PR-3 in the presence of EGCG
The proteolytic activity of PR-3 on gelatin was investigated using the gelatin-zymography assay. Figure 1 shows that 2 µg/60 ml PR-3 produced a major gelatinolytic band of approximately 29 kDa (first lane on the left of the inset in Fig. 1A ); a minor band of lower molecular weight (very probably corresponding to the residual zymogen form of 35 kDa) and one of higher molecular weight were also generated. When the zymography was developed in the presence of an increasing concentration of EGCG (0–250 µM), the intensity of the digestion bands progressively decreased and completely disappeared at the highest concentration. The gelatinolytic activity of the 29-kDa band appeared to be the most inhibited by EGCG, and densitometric analysis indicated an IC50 < 20 µM (Fig. 1A) .



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  		Figure 1. (A) The gelatinolytic activity of PR-3 is inhibited by EGCG in a dose-dependent manner. The gelatin zymogram (2 µg PR-3) was developed in the presence of increasing concentrations of EGCG (inset) and quantitated by densitometric analysis (graph); example of triplicate experiment, run in 10–24% polyacrylamide gel. (B) EGCG (100 µM) restrains degradation of casein–FITC by PR-3. (C) EGCG (100 µM) does not impair synthetic substrate [MeO-Suc-K(Pic)APV-pNA] degradation by PR-3. (B and C) Time-course reactions of triplicate experiment; each point represents average of triplicates (SD<15%). O.D., Optical density.

 
The proteolytic activity of PR-3 on casein was restrained, although to a lesser extent, in a dose-dependent manner (not shown) by the presence of EGCG, and 100 µM gave approximately 50% inhibition (Fig. 1B) .

Conversely, when the MeO-Suc-K(Pic)APV-pNA synthetic substrate, suitable for PR-3 and LE [31 ], was used, EGCG exerted a very marginal effect on the lytic activity of PR-3, even at the highest concentration compatible with the colorimetric reaction (100 µM; Fig. 1C ). When PR-3 was incubated with MeO-Suc-AAPV-pNA, substrate of election for elastase, some inhibition was registered after 3 h: -10%, -15%, -30%, at 1, 10, and 100 µM EGCG, respectively (not shown).

Dose-dependent activation of pro-MMP-2 by PR-3
First, HT1080 human fibrosarcoma cells were incubated in the presence or absence of PR-3 for 2 h, then the conditioned medium and the cell membrane extracts were analyzed by gelatin zymography. This revealed that although the pro-MMP-2 in the former underwent activation (Fig. 2A ), the latter failed to be processed (Fig. 2B) . Then, to further study the activating potential of PR-3 on MMP-2, a pro-MMP-2-containing culture medium was used with no trace of the MMP-2-activated form: After preincubation of this medium in the presence of NEM (cysteine-proteinase inhibitor) and an increasing amount of PR-3 (0–50 µg/ml), gelatin zymography revealed a progressive decrease of the MMP-2 zymogen and increase of the activated form; the latter was already clearly visible with 50 ng/ml PR-3 (Fig. 2C) .



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  		Figure 2. (A and B) PR-3-triggered activation of pro-MMP-2 in cell culture. (A) Lanes were loaded with medium conditioned by 5 x 104 HT1080 cells incubated 2 h in the presence of PR-3; (B) lanes with membrane extract from 2 x 107 HT1080 cells in the same conditions. (C) Cell-free, dose-dependent activation of pro-MMP-2 in solution by PR-3; the MMP-2/PR-3 mixture was preincubated 5 h at 37°C, and equal volumes were analyzed by gelatin zymography. Comparison of B with A shows lack of activation of pro-MMP-2 when bound to the cell membrane versus that secreted into the medium. The zymogen form (z) of the latter decreases, and its activated form (a) increases from 0.05 to 50 µg/ml PR-3 (C). Gelatin zymograms with 9% (A and B) and 6% (C) polyacrylamide. Example of triplicate experiment.

 
Activation of pro-MMP-2 by PMNs and influence of exogenous PR-3 on tumor cell invasion in vitro
The potential activation of pro-MMP-2 triggered by the presence of activated PMNs (non-MMP-2-producing cells) was investigated by coincubating PMNs with HT1080 cells in a 1:1 and 10:1 ratio for 2 and 4 h after removal of PMNs by mechanical shock before analysis. Gelatin zymography of the conditioned medium after 4 h revealed that the zymogen is partially converted to the activated form (Fig. 3A ). Instead, at cell-membrane level, although addition of PMNs 1:1 shows a limited level of pro-MMP-2 activation, addition of PMNs 10:1 almost completely eliminates MMP-2 activity in the zymogen and activated form (Fig. 3B) ; the same result was already registered after 2 h.



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  		Figure 3. (A) Addition of PMNs to HT1080 human fibrosarcoma cells triggers activation of secreted pro-MMP-2 and (B) degrades membrane-bound MMP-2 (lane 10:1). HT1080 cells (107) were incubated 4 h at 37°C with PMNs (107 in lane 1:1, 108 in lane 10:1), and the clarified media (conditioned by 5x104 HT1080 cells) and the membrane extracts (total) were analyzed by gelatin zymography. Example of a triplicate experiment run in 8% polyacrylamide gel. (C) Invasion of HT1080 cells through MatrigelTM is inhibited by addition of exogenous PR-3 in a dose-dependent manner. Example of triplicate experiment, and each point represents average of sixtuplicates (SD<15%).

 
In conditioned medium and membrane extract of cocultures, two major bands of gelatinolysis are present, representing the zymogen and activated forms of MMP-9 contributed by PMNs. To study whether this proteinase derives from the load of residual PMNs (mechanically removed after coincubation) or else from the enzyme released by PMNs and captured by tumor cells, clarified MMP-9-containing medium (conditioned by PMNs) was added to HT1080 cells: The zymogram of the membrane extract after incubation did not reveal MMP-9 activity (not shown).

Given the lytic activity exerted by PR-3 on several ECM proteins, including basement membrane components, and its potential to directly activate two gelatinases instrumental in cellular invasion, we assayed the influence of PR-3 on tumor cell invasion through a reconstituted basement membrane (MatrigelTM). In a modified Boyden-chamber assay using filters coated with gelatin and MatrigelTM, the presence of PR-3 (0–6 µg/ml) in the upper chamber (where HT1080 cells are seeded at T0) significantly reduced tumor cell invasion in a dose-dependent manner (Fig. 3C) . HT1080 invasion was also inhibited (by ~40%) when PR-3 was substituted with 5 x 104 PMNs (not shown).

MMP-2 activation by three neutrophil serine-proteases
Gelatin-zymographic analysis of pro-MMP-2-containing medium, preincubated with the three main neutrophil-serine-proteases, single or in combination, revealed that the propeptide of the MMP-2 zymogen is also cleaved by 50 mU/ml Cat.G (Fig. 4 , lane 4) with an efficacy similar to PR-3 (Fig. 4 , lane 2). Conversely, the same amount of LE had no appreciable effect on the activation (Fig. 4 , lane 3). The combination of the two active proteases PR-3 with Cat.G, but also of PR-3 with LE, slightly enhances MMP-2 activation, as did a combination of the three enzymes.



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  		Figure 4. MMP-2 activation by three serine-proteases, single or in combination. PR-3 (50 µg/ml, lane 2) and Cat.G (50 mU/ml, lane 4) individually activate MMP-2 [zymogen (z)>activated (a) form], but incubation of pro-MMP-2 with both together (lane 6) does not result in the sum of separate activations. LE (50 mU/ml, lane 3) fails to activate pro-MMP-2. Example of triplicate experiment run in 8% polyacrylamide gel.

 
Activation of MMP-2 and MMP-9 by PR-3 in the presence of EGCG
A 10–24% gradient polyacrylamide gelatin zymography was used to give a preliminary check, which under the given, nondenaturing conditions, pro-MMP-2 and PR-3 could be separated into distinct gelatinolytic bands, as shown in Figure 5A . The effect of PR-3 on MMP-2 and MMP-9 zymogen forms in the presence of EGCG was then analyzed by gelatin zymography: PR-3 was able to activate MMP-2 and MMP-9, and this was not affected by 5 and 15 µM EGCG (10 and 30 times the physiologically reachable serum concentration in green tea drinkers; Fig. 5B ). Incubation of gelatinase-containing medium with EGCG alone did not alter the control bands.



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  		Figure 5. (A) Gelatinolytic pattern of pro-MMP-2 and PR-3 (2 µg) in 10–24% polyacrylamide gel. (B) Activation of pro-MMP-2 and pro-MMP-9 by PR-3 (50 µg/ml) is not inhibited by EGCG. PR-3 was first incubated 30 min at 4°C with EGCG, then added to pro-MMP-containing medium, and incubated 5 h at 37°C before electrophoresis (z=zymogen; a=activated form). Example of triplicate experiment run in 6% polyacrylamide gel.

 
Degradation of MatrigelTM by PR-3 or PMNs in the presence of EGCG
PR-3 activity on a reconstituted basement membrane matrix (MatrigelTM) was also investigated. After 3 h incubation at 37°C with gelified MatrigelTM (0.84 mg/ml), 1 µg/ml PR-3 produced clear degradation of laminin (400 and 220 kDa chains) and type IV collagen (Fig. 6 , lane 2 vs. lane 1); conversely, MatrigelTM components were well preserved in a dose-dependent manner when EGCG (0–60 µM) was added to the mixture. Figure 6 , lanes 1–3, shows an example at 15 µM EGCG. Similar results were obtained substituting PR-3 with PMNs for 5 h (Fig. 6 , lanes 4–6), but in this case, a marginal degradation of laminin was observed.



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  		Figure 6. (A) Degradation of MatrigelTM components [L400 and L220 chains of laminin and type IV collagen (cIV)] by PR-3 or PMNs is inhibited by EGCG. Gelified MatrigelTM (50 µl of 0.8 mg/ml) was incubated with 30 µl of 6.7 µg/ml PR-3 (lanes 2 and 3) or 1.2 x 105 PMNs for 3 or 5 h, respectively, in 200 µl final volume (lanes 5 and 6) in the presence or absence of the catechin for 5 h, respectively, at 37°C. Example of duplicate experiment run in 5% polyacrylamide gel.

 
Immunolocalization of PR-3 and MMP-2 in the presence of EGCG
Our study also investigated whether PR-3 binds to cell membrane and colocalizes with MMP-2 and whether its localization is affected by the presence of EGCG. When exogenous PR-3 was added to HT1080 human fibrosarcoma cells, immunolocalization revealed retention of the protease around the entire cell membrane and in particular, colocalization (merge) with MMP-2 in a few limited areas (Fig. 7 ). The presence of 10 µM EGCG did not result in appreciable modifications in the cellular localization and/or colocalization of PR-3 and MMP-2.



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  		Figure 7. Colocalization of PR-3 and MMP-2 on cell membrane is not modified by EGCG. HT1080 human fibrosarcoma cell cultures were incubated 30 min at 4°C in the presence of PR-3 (30 µg/ml); then, EGCG (10 µM) was added to the medium and incubated 60 min at 4°C. The two proteases were immunodetected by the use of specific antibodies. Merge shows colocalization in yellow; example of duplicate experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Gelatin zymography of the commercially available PR-3 reveals three gelatinolytic bands (Figs. 1A and 5A) : a major one and two minor ones of higher and lower molecular weight corresponding to the 29-kDa activated form, the COOH-terminal-truncated form, and the 35-kDa zymogen of the enzyme, respectively [8 ]. It is worth noticing that following SDS-PAGE, a zymogen may exert enzymatic activity: This has been reported for other pro-gelatinases when assayed by zymography [24 , 32 ] and explained as the nonproteolytic exposure of the active site, following SDS and trapping into the gel.

PR-3 can thus be added as a new member to the numerous gelatinase families, which already include metallo-proteinases (MMP-2, MMP-9, macrophage metallo-elastase, collagenase-3, MT-MMPs, gelatinase C, and meprin), serine-proteinases (LE, guanidino-benzoatase, seprase, and thrombin), and one cysteine-proteinase (cysteine-like gelatinase).

Various roles have been described for PR-3 in a number of physiological and pathological conditions, as already recalled in Introduction, where this proteinase is instrumental to the lysis of normal tissue architecture. The newly described gelatinolytic activity of PR-3 now suggests that this proteinase may contribute to the efficient removal of denatured collagen following the acute phase of tissue damage.

In addition to this "scavenging" activity, PR-3 may play an active role in the removal of the propeptide of in-solution MMP-2 and MMP-9 (Figs. 2C 4 and 5B) , whose action degrades basement membrane collagen and other ECM proteins [11 ]. It has already been demonstrated that direct activation of pro-MMP-9 may be achieved as efficiently by PR-3 as by LE but less so than by Cat.G and much less than by stromelysin-1 (MMP-3) [12 ]. We now show that PR-3 has the potential to activate in-solution pro-MMP-2 and pro-MMP-9 in a dose-dependent manner and that this activation may be triggered also by PMNs coincubated with MMP-2 zymogen-secreting cells (HT1080): In the latter case, Cat.G but not neutrophil elastase (here and ref. [26 ]) may contribute to the direct activation of pro-MMP-2, as demonstrated by biochemical assays with purified enzymes (Fig. 4) .

Regarding pro-MMP-2 activation, results from incubation of pro-MMP-2-producing cells in the presence of PR-3 recently suggested a concerted action between PR-3 and MT1-MMP, a membrane-bound activator of pro-MMP-2, thus excluding a direct activation of the gelatinase by PR-3 alone [13 ]. In a cell-free system, the use of commercially available, highly purified PR-3 now shows that this enzyme has the potential to activate in-solution pro-MMP-2 and pro-MMP-9 without the cooperation of MT1-MMP (Figs. 2C 4 and 5B) . The latter is in fact a metallo-proteinase, which in the case of autocatalytic shedding, terminates its activity [33 ]; thus, in the pro-MMP-2-containing medium, used here for the activation experiments, the presence of proteolytically active MT1-MMP, which may cooperate with PR-3, should be excluded.

Moreover, the hypothesis that the membrane-bound MT1-MMP may render the activation of the gelatinase by other proteinases including PR-3 more efficient and focused by blocking pro-MMP-2 at the cell surface [11 ] should be reconsidered (at least for PR-3). In fact, membrane-bound MMP-2 is scantly activated or fully degraded following incubation of PMNs with HT1080 cells, in 1:1 and 10:1 ratios, respectively (Fig. 3B) , and addition of purified PR-3 to HT1080 cells leaves unchanged membrane-bound pro-MMP-2 (Fig. 2B) ; in the former case, the registered elimination of MMP-2 must then be triggered by other neutrophil proteinases.

Nevertheless, although MMP-2 is wiped out by the intervention of PMNs, under the same conditions, a large amount of MMP-9 of PMN origin is recovered after PMN removal, along with the HT1080 cell membrane extract. As in the conditions used MMP-9 is not expressed by HT1080 cells, account must be taken of a possible contribution by inflammatory cells to membrane-bound, tumor proteolytic machinery instrumental to invasion and metastasis [34 ]. Following incubation of HT1080 cells with medium alone conditioned by PMNs, binding of MMP-9 to HT1080 membrane was not evident; the MMP-9 detected after coincubation has thus to be attributed to residual (post-rinsing) PMNs present in the sample, therefore excluding tumor-cell capture of MMP-9 secreted by inflammatory cells.

It has been suggested that a specific, 111-kDa membrane molecule binds the neutrophil-released PR-3 at the surface of other cells [35 ], and it may indeed be involved in the rapid binding of PR-3 at the cell surface of invasive tumor cells. This binding is now shown by immunolocalization of exogenous proteinase and does not seem to be affected by EGCG, the main flavanol present in green tea. As for other proteinases (MMP-2/MT1-MMP) [25 ], micromolar concentration of EGCG does not show appreciable impairment of PR-3 binding to the cell membrane as well as PR-3/MMP-2 colocalization (Fig. 7) ; the latter aspect needs further investigation anyway (Fig. 7) .

Furthermore, the registered PR-3 potential of activating in-solution pro-gelatinases was not impaired by EGCG at concentrations (15 µM) that instead give 50% inhibition of MMP-2 and MMP-9 activity [24 , 28 ]. Together with the fact that EGCG (100 µM) was ineffective also in inhibiting degradation of PR-3 synthetic substrate, while exerting inhibition of casein degradation, this suggests that the flavanol may interfere with portions of natural substrates not represented in the synthetic peptide and with portions of PR-3 not involved in substrate binding and its active site. On the contrary, degradation of MatrigelTM components (type IV collagen and laminin) by PR-3 was clearly hindered by 15 µM EGCG (Fig. 6) . The molecular mechanisms behind this differential disturbance need further investigation.

Intriguingly, when tumor cells (HT1080) were tested in a Boyden chamber under chemotactic stimulus in the presence of PR-3 or PMNs, a dose-dependent restraint of the invasive behavior through MatrigelTM was clearly registered. After incubation with PR-3 or PMNs, analysis of MatrigelTM molecules shows that the main structural component, type IV collagen, is completely degraded, and laminin is also affected (Fig. 6) . A proteolytic load lower in PR-3 and other enzymes very likely accounts for the lower degradation of laminin registered with PMNs. In any case, the degradation of basement membrane components or adhesion molecules [6 7 8 ], impairing the anchorage essential for cell migration, may explain the reduced invasion registered. The effect of EGCG in this model system, which to date, has not given clear-cut results (not shown), requires further investigation.

Conversely, EGCG was able to restrain PR-3 gelatinolytic activity in a dose-dependent manner, with an IC50 < 20 µM, similar to that registered for MMP-2, MMP-9, and MT-MMP. It is worth mentioning that among serine-proteinases, this inhibition is much higher than that on Cat.G (IC50=1 mM) but lower than that exerted by EGCG on LE (IC50=0.3 µM) [26 ]. Compared with the latter, the inhibition of the PR-3 activity on a synthetic elastin peptide is also much lower. This differential, inhibitory efficacy on proteinases instrumental in ECM protein degradation may thus prove to be advantageous for clinical use of EGCG in humans when reactions mediated by Cat.G or a urokinase-type plasminogen activator need to be preserved.

In conclusion, PR-3 is the second serine-proteinase after plasmin [36 ] shown to be able to activate pro-MMP-2 directly; PR-3 is able to degrade gelatin, laminin, and type IV collagen in a reconstituted basement membrane structure; and this degradation is inhibited by EGCG. Whether and to what extent this inhibition may contribute to the already reported anti-inflammatory activity of EGCG [26 , 27 ], and activated in-solution gelatinases interact with ECM molecules deserve further investigation. Regarding cancer invasion, the evidence that direct degradation of basement membrane components by PR-3 is restrained by the flavanol prompts further investigation of the possibility of improving inhibition of proteolytic activities triggered by inflammatory events and potentially instrumental in cancer aggressiveness.


   ACKNOWLEDGEMENTS
 
This research was supported by grants from the "Associazione Italiana per la Ricerca sul Cancro" (Milan) and from the MIUR (Rome) of the Italian government. We are grateful to Dr. G. De Silvestro (Immuno-Transfusion Service, Padova Hospital, Italy) for supplying buffy coats and Dr. S. Biggin for revision of the English manuscript.

Received February 28, 2003; revised March 7, 2003; accepted March 17, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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