©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Matrix Metalloproteinase 7 (Matrilysin) from Human Rectal Carcinoma Cells
ACTIVATION OF THE PRECURSOR, INTERACTION WITH OTHER MATRIX METALLOPROTEINASES AND ENZYMIC PROPERTIES (*)

(Received for publication, October 21, 1994; and in revised form, January 18, 1995)

Kazushi Imai (1) (3) Yasuo Yokohama (2) Isao Nakanishi (3) Eiko Ohuchi (4) Yutaka Fujii (5) Noboru Nakai (5) Yasunori Okada (1)(§)

From the  (1)Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, Kanazawa 920, Departments of (2)Orthopedic Surgery and (3)Pathology, School of Medicine, Kanazawa University, Kanazawa 920, the (4)Fuji Chemical Industries, Ltd., Takaoka 933, and the (5)Department of Chemistry, Fukui Medical School, Fukui 910-11, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Matrix metalloproteinase 7 (MMP-7) has been purified as an inactive zymogen of M(r) 28,000 (proMMP-7) from the culture medium of CaR-1 human rectal carcinoma cells. The NH(2)-terminal sequence of proMMP-7 is Lys-Pro-Lys-Pro-Gln-Glu, which is identical to that of matrilysin. The zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding an intermediate form of M(r) 21,000 and an active species of M(r) 19,000 which shows the new NH(2)-terminal sequence of Tyr-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully activates the zymogen, the activation rate by plasmin or leukocyte elastase is confined to 50%. ProMMP-7 can be activated by MMP-3 (stromelysin 1) to its full activity in a single-step mechanism and generates the same NH(2) terminus obtained by APMA activation, whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9 (gelatinase B) do not have such an effect. On the other hand, proMMP-1 is activated by MMP-7 to an activity similar to that obtained by APMA and the activation by MMP-7 is enhanced up to 6.5-fold in the presence of APMA. This enhanced activity is donated by specific cleavage at the Gln-Phe bond of proMMP-1. MMP-7 can also activate proMMP-9 up to 50% of the full activity with a new NH(2) terminus of Leu-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3 with MMP-7 results in no activation of these proMMPs. MMP-7 degrades type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin, and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix macromolecules in concert with MMP-1, -3, and -9 under pathological conditions.


INTRODUCTION

The matrix metalloproteinases (MMPs) (^1)are a gene family of Zn endopeptidases that can digest various extracellular matrix macromolecules. Ten members have been identified and classified into four major types of structurally and functionally related metalloproteinases: 1) interstitial collagenases (tissue collagenase = MMP-1; neutrophil collagenase = MMP-8; collagenase 3 = MMP-13), which degrade fibrillar collagens; 2) gelatinases/type IV collagenases (72-kDa metalloproteinase = gelatinase A = MMP-2; 92-kDa metalloproteinase = gelatinase B = MMP-9), which hydrolyze gelatins and type IV and V collagens; 3) stromelysins (stromelysin 1 = MMP-3; stromelysin 2 = MMP-10), which are active against a wide range of substrates including proteoglycans, laminin, fibronectin, type IV collagen, and telopeptides of other collagens; 4) other MMPs (matrilysin = MMP-7; stromelysin 3 = MMP-11; macrophage metalloelastase = MMP-12)(1, 2) . Recent studies on MMP-11 have demonstrated a weak activity against fibronectin, laminin, proteoglycan, and gelatin(3) . MMP-12 is known to digest insoluble elastin although no information is available about the activity against other extracellular matrix macromolecules(4) . MMP-7 is unique in size for lack of the COOH-terminal domain. The proteinase can cleave a wide range of substrates such as fibronectin, laminin, proteoglycan, elastin, gelatin, type IV collagen, aggrecan, and entactin(5, 6, 7, 8, 9, 10) , and the substrate specificity is reported to be similar to that of MMP-3 (8) . It has been shown that the zymogen of MMP-7 (proMMP-7) is activated by 4-aminophenylmercuric acetate (APMA) and trypsin following the stepwise mechanisms proposed for other proMMPs such as proMMP-3 (11) . However, little is known about activation by other factors including serine proteinases and active oxygen metabolites which are capable of activating many proMMPs and are present in pathological conditions(1) . A growing body of evidence has disclosed intermolecular activation of proMMPs. It is well known that MMP-3 is an effective activator for proMMP-1, -8, and -9(12, 13, 14, 15, 16) . Suzuki et al.(13) have examined the precise mechanisms of proMMP-1 activation and demonstrated that MMP-3 fully activates proMMP-1 by cleavage of the Gln-Phe bond of the proenzyme. Although similar activation of proMMP-1 is reported to occur with MMP-7(8) , no studies have been made on the molecular mechanisms. In fact, very limited information about the interactions of MMP-7 with other MMPs is available.

In this study, we have purified proMMP-7 from the culture medium of CaR-1 human rectal carcinoma cells and studied the activation of the zymogen, interactions with other MMPs, and the enzymic properties. The present studies demonstrate that proMMP-7 is activated by MMP-3 to its full activity, and conversely MMP-7 can activate proMMP-1 and proMMP-9. We also show that MMP-7 has a wide range of substrate specificity, which is different from that of MMP-3 in specific activity.


EXPERIMENTAL PROCEDURES

Materials

Materials were obtained as follows: alpha-chymotrypsin, cysteine, diisopropyl fluorophosphate (DIFP), EP 475, N-ethylmaleimide, leupeptin, 2-mercaptoethanol, [l-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin (bovine pancreas), pepstatin A, 1,10-phenanthroline, phenylmethanesulfonyl fluoride, plasma kallikrein, phosphoramidon, plasminogen (human), soya-bean trypsin inhibitor, and thrombin (bovine) from Sigma; 1,4-dithiothreitol and iodoacetamide from Wako Chem., Japan; alpha(2)-macroglobulin from Boehringer Mannheim GmbH, Germany; cathepsin G from ICN Biochemicals (Cleveland, OH); Green A Dyematrex gel from Amicon Corp. (Beverly, MA); Chelating Sepharose Fast Flow, Sephadex G-10, and Ultrogel AcA 44 from Pharmacia Fine Chemicals, Sweden; DEAE-cellulose (DE52) from Whatman, United Kingdom. ProMMP-1, -2, -3, and -9, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were purified as described previously(16, 17, 18, 19, 20, 21) . Laminin-1 and type IV collagen were purified from Engelbreth-Holm-Swarm tumor(22, 23) , type III, V, and VI collagens from human placenta(24, 25) , and fibronectin from human plasma(26) . Type II collagen from bovine cartilage was purchased from Nitta Gelatin (Japan) and insoluble elastin from Elastin Products Co. (Owensville, MO). Bovine nasal cartilage proteoglycan subunit was prepared according to the method of Nagase and Woessner(27) .

Enzyme Purification

ProMMP-7 was purified by a five-step protocol from culture medium of CaR-1 human rectal carcinoma cells, which were obtained from the Japanese Cancer Research Resource Cell Bank. The first two steps used for the purification of proMMP-7 were essentially the same as those reported for the purification of proMMP-3 and proMMP-9(16, 18) . The concentrated culture medium was first applied to a column of DEAE-cellulose (2.5 times 8.5 cm), and the unbound fractions were subjected to a column of Green A Dyematrex gel (2.5 times 8 cm). ProMMP-7 was eluted by a linear gradient of NaCl (0.15 M-2 M), dialyzed against 10 mM Tris-HCl, pH 8.0, 5 mM CaCl(2), 0.05% Brij 35, 0.02% NaN(3), and applied to a column of DEAE-cellulose (1 times 18.5 cm) equilibrated with the same buffer. The unbound fractions was dialyzed against 25 mM sodium borate buffer, pH 8.0, 0.15 M NaCl, 1 mM CaCl(2), 0.05% Brij 35, 0.02% NaN(3) and subjected to a column of Chelating Sepharose Fast Flow (0.7 times 8 cm) charging with 0.2 M ZnCl(2). Active MMP-7 was eluted with 0.15 M NaCl and proMMP-7 with 1 M NaCl in 25 mM sodium cacodylate buffer, pH 6.5, 1 mM CaCl(2), 0.05% Brij 35, and 0.02% NaN(3). Fractions containing proMMP-7 were chromatographed on a column of Ultrogel AcA 44 (1.5 times 115 cm) as previously reported(18) . Fractions containing a single protein band of M(r) 28,000 on SDS-PAGE were combined and used as the source of pure proMMP-7.

Enzyme Assays

^14C-Acetylated type I collagen and its heat-denatured collagen (gelatin) were used for the assays to measure the activities of MMP-1 (28) and MMP-2 and -9(16, 17) , respectively. The enzymic activities of MMP-3 and MMP-7 were determined using ^3H-labeled carboxymethylated transferrin (Cm-Tf) (29) . APMA-activated MMP-1, -2, -3, and -9 were prepared (16, 17, 18, 19) and subjected to spin columns to remove APMA(29) . Based on molecular masses of 51,929, 70,952, 53,997, 27,938, and 78,426 for MMP-1, -2, -3, -7, and -9, the extinction coefficients were calculated and E = 1.3, 1.9, 1.1, 1.6, and 1.3 ml/mg for MMP-1, -2, -3, -7, and -9, respectively, were used for the studies. Activation studies of proMMP-7 by active oxygen metabolites and acid exposure were performed according to our previous methods(16, 30) . For activation studies, the activation rate was calculated by taking the activities of APMA-activated MMPs as 100% activity. The activities of MMPs to cartilage proteoglycan and insoluble elastin were assayed as described previously(27, 31) .

Sequence Analyses

The NH(2)-terminal sequence of proMMP-7 and APMA-activated MMP-7 was determined by an Applied Biosystems 477A Protein Sequencer with on-line high performance liquid chromatography (Applied Biosystems, Foster, CA). For the sequence analyses of active MMP species prepared by intermolecular activation, the enzyme species were separated by SDS-PAGE under the reducing conditions and transferred to polyvinylidene difluoride membrane(32) . The proteins on the membrane were located by staining with 0.1% Amido Black 10B. The bands of interest were excised and sequenced.

Immunoblot Analyses

Samples resolved by SDS-PAGE with reduction were transferred onto nitrocellulose filters. The filters were reacted with monoclonal antibodies, and protein bands were visualized by avidin-biotin-peroxidase complex method as described previously(16) . The monoclonal antibodies specific to NH(2)-terminal (residues 31-49) or COOH-terminal domain (residues 643-661) of proMMP-9 and that to proMMP-3 were prepared and characterized previously(16, 33) . Monoclonal antibody against matrilysin was developed using a synthetic peptide corresponding to the amino acid sequence of the COOHterminal peptide of human matrilysin (residues 253-267, GIQKLYGKRSNSRKK)(34) .

Digestion of Extracellular Matrix Macromolecules

ProMMP-7 was maximally activated by reaction with 1 mM APMA for 4 h at 37 °C and incubated with various protein substrates in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl(2), 0.05% Brij 35, 0.02% NaN(3) at the indicated temperatures. The reaction was stopped with 20 mM EDTA and products were analyzed by SDS-PAGE.


RESULTS

Purification and Properties of ProMMP-7

The purified proMMP-7 was homogeneous on SDS-PAGE showing M(r) 28,000 under both reducing and non-reducing conditions and had an NH(2) terminus of Lys-Pro-Lys-Pro-Gln-Glu. Both proMMP-7 in the culture medium and purified proMMP-7 were recognized by a peptide-specific monoclonal antibody to matrilysin (clone 125-20H11) (data not shown). The culture medium contained no protein bands recognizable by the antibody to MMP-3 (data not shown). The final product exhibited a specific activity of 8,095 µg digestion/min/mg of enzyme at 37 °C against [^3H]Cm-Tf when assayed after activation with 1 mM APMA for 4 h at 37 °C. The overall yield of proMMP-7 was 46% with 193-fold purification after the first DEAE-cellulose step.

APMA-activated MMP-7 was stable without loss of the activity even after storage at 23 °C for 4 weeks, although proMMP-7 showed spontaneous activation (up to 30%) in the same condition. However, there was no decrease in the activity or spontaneous activation of proMMP-7 when stored at 4 °C for the same period. On the other hand, proMMP-7 became the active species of M(r) 19,000 when thawed after storage for a month at -20 °C. Inhibitor studies indicated that MMP-7 is a typical metalloproteinase. The activity of MMP-7 was completely inhibited by chelating agents including EDTA, EGTA, and 1,10-phenanthroline, TIMP-1 and TIMP-2, and thiol compounds (data not shown). SDS and 2-mercaptoethanol were also inhibitory. However, inhibitors of serine, cysteine, or aspartic proteinases did not show any significant effect.

Activation of ProMMP-7

Time Course Activation of ProMMP-7 by APMA

The activation of proMMP-7 with 1 mM APMA was slower than that of proMMP-2 (17) and faster than that of proMMP-3 or -9(16, 18) : 30 min of incubation at 37 °C gave 65% of the full activity and maximal activation was obtained after 4 h-incubation (Fig. 1A). Prolonged incubation reduced the activity but 80% of the activity remained even after 24 h of incubation. A similar activation curve was obtained with 0.25, 0.5, and 0.75 mM APMA. ProMMP-7 was also activated to its full activity even with 0.1 mM APMA, although it took a longer (8 h) incubation for full activation. However, incubation with 1.5 mM APMA resulted in only about 70% activation of proMMP-7 (data not shown).


Figure 1: Time course of proMMP-7 activation by APMA and conversion of proMMP-7 during the activation. A, proMMP-7 (55 ng) was incubated with (bullet) or without (circle) 1 mM APMA for up to 24 h at 37 °C. At the incubation times indicated, the samples were subjected to the assay using [^3H]Cm-Tf for 30 min at 37 °C. B, ProMMP-7 (700 ng) incubated with 1 mM APMA was also analyzed by SDS-PAGE (12.5% total acrylamide) under reducing conditions. After electrophoresis the gel was stained with silver nitrate. C0 and C24, proMMP-7 incubated at 37 °C with buffer alone for 0 and 24 h, respectively. Protein standards are phosphorylase b (94 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soya-bean trypsin inhibitor (21 kDa), and lysozyme (14 kDa).



During activation with 1 mM APMA, proMMP-7 of M(r) 28,000 was processed initially to a molecule of M(r) 21,000 and then converted to the M(r) 19,000 species (Fig. 1B). When these MMP-7 species were compared with the activation curve, generation of the M(r) 19,000 species was correlated with the activity, indicating that the M(r) 19,000 form is an active species. The NH(2) terminus of the active species of MMP-7 was Tyr-Ser-Leu-Phe-Pro-Asn-Ser.

Activation of ProMMP-7 by Endopeptidases and Other Agents

Seven different serine proteinases including trypsin, alpha-chymotrypsin, plasmin, leukocyte elastase, cathepsin G, thrombin, and plasma kallikrein were examined for their ability to activate proMMP-7. Fig. 2shows that trypsin (1 µg/ml) can activate proMMP-7 to 95% of the full activity after 8 h of incubation at 37 °C. At a higher concentration of trypsin (10 µg/ml), the zymogen was rapidly activated up to 85% after 8 h of incubation (Fig. 2), and the activity declined by further incubation up to 24 h (data not shown). In order to study the action of trypsin on proMMP-7 by SDS-PAGE I-labeled proMMP-7 was used, since radioiodination did not cause any significant changes in the intrinsic properties of the zymogen. Incubation of I-labeled proMMP-7 with trypsin (1 µg/ml) converted the precursor first into a fragment of M(r) 22,500, which was then processed to a doublet of M(r) 21,000 and 20,000 species after 8 h of incubation (Fig. 2). Contrary to our expectation, proMMP-7 was not activated at all with alpha-chymotrypsin (0.1, 1, and 10 µg/ml), but the zymogen was degraded to a M(r) 15,500 species with 10 µg/ml alpha-chymotrypsin (data not shown). Plasmin (10 µg/ml) and leukocyte elastase (10 µg/ml) could also activate proMMP-7 up to 55 and 45% of the full activity, respectively, although at lower concentrations (0.1 and 1 µg/ml) no significant activation was observed (Fig. 3). During incubation with these serine proteinases (10 µg/ml), proMMP-7 was processed to a protein band of M(r) 19,000 with plasmin and that of M(r) 20,500 with leukocyte elastase (Fig. 3). Cathepsin G itself was a poor activator for proMMP-7, giving only 20% of the full activity with conversion of the precursor into M(r) 22,000 and 20,500 forms after 8 h of incubation at 37 °C. However, the proteinase accelerated the proMMP-7 activation process by plasmin or leukocyte elastase without changing the activation rate (Fig. 3). Thrombin and plasma kallikrein did not activate proMMP-7 at all.


Figure 2: Time course activation and conversion of proMMP-7 by trypsin. ProMMP-7 (55 ng) was incubated with trypsin at 37 °C at 10 µg/ml (up triangle), 1 µg/ml (box), 0.1 µg/ml (circle), and 0 µg/ml (times). The MMP-7 activity was measured against [^3H]Cm-Tf for 30 min at 37 °C after termination of the reaction with 2 mM DIFP. Activity achieved by incubation of proMMP-7 with 1 mM APMA for 4 h at 37 °C was taken as 100% activity. Inset, conversion of proMMP-7 by trypsin activation. A mixture of I-labeled and unlabeled proMMP-7 (55 ng) incubated with trypsin (1 µg/ml) up to 8 h was electrophoresed on SDS (12.5% total acrylamide) gel. After electrophoresis the gel was dried and autoradiographed. Upper arrow, proMMP-7 of M(r) 28,000; middle arrow, a species of MMP-7 with M(r) 22,500; lower arrow, a doublet of MMP-7 forms with M(r) 21,000 and 20,000. C0, proMMP-7 incubated with buffer alone for 0 h at 37 °C.




Figure 3: Activation of proMMP-7 by plasmin or leukocyte elastase in the presence or absence of cathepsin G. ProMMP-7 (55 ng) was treated with plasmin (A) or leukocyte elastase (B) at 37 °C at 10 µg/ml (box), 1 µg/ml (up triangle), or 0.1 µg/ml (circle) in the absence of cathepsin G, and at 10 µg/ml in the presence of 10 µg/ml cathepsin G (times). The reaction was stopped with 2 mM DIFP, and MMP-7 activity was assayed against [^3H]Cm-Tf for 30 min at 37 °C. Activity obtained by incubation of proMMP-7 with 1 mM APMA for 4 h at 37 °C was taken as 100% activity. No activation occurred with proMMP-7 incubated with buffer alone up to 8 h (data not shown). Inset, M(r) changes in proMMP-7 during activation with 10 µg/ml plasmin (A) or 10 µg/ml leukocyte elastase (B). The reaction products were analyzed on SDS-PAGE as described in Fig. 2. A: upper arrow, proMMP-7 of M(r) 28,000; lower arrow, a species of M(r) 19,000. B: upper arrow, proMMP-7 of M(r) 28,000; lower arrow, a species of M(r) 20,500. C0, proMMP-7 incubated with buffer alone for 0 h.



To examine the effect of active oxygen metabolites on proMMP-7 activation, the zymogen was reacted with various concentrations of HOCl or H(2)O(2). However, both reagents did not activate proMMP-7 (data not shown). The possibility of proMMP-7 activation by acid exposure, another activation mechanism shown with proMMP-9(30, 35) , was also examined by incubation of the zymogen in the range from 2.4 to 9.7. However, no activation occurred by the treatment (data not shown).

Interaction of MMP-7 with MMP-1, -2, -3, and -9

Activation of ProMMP-7 by MMP-1, 2, 3, and 9

Previous studies have demonstrated that MMP-3 is an effective activator for proMMP-1 (12, 13) , proMMP-8(14) , and proMMP-9(15, 16) . Therefore, we examined whether MMP-3 can activate proMMP-7. As shown in Fig. 4A, proMMP-7 was fully activated by MMP-3 in a dose-dependent manner. The activation was dependent on MMP-3 activity since neither MMP-3 inactivated by heat or 1,10-phenanthroline treatment nor proMMP-3 could activate proMMP-7. During the activation with MMP-3 proMMP-7 was processed to an active species with M(r) 19,000 without any intermediate form on SDS-PAGE (Fig. 4B). In the presence of 1 mM APMA, MMP-3 accelerated proMMP-7 activation in a dose-dependent manner (Fig. 4A) and proMMP-7 was rapidly processed to an active species with M(r) 19,000 (data not shown). NH(2)-terminal sequence analysis of MMP-7 activated by MMP-3 showed the sequence of Tyr-Ser-Leu-Phe-Pro, which is identical to that of APMA-activated MMP-7. In contrast to proMMP-7 activation by MMP-3, none of MMP-1, 2, and 9 activated proMMP-7.


Figure 4: Activation of proMMP-7 by MMP-3. A, proMMP-7 (550 ng) was incubated with active MMP-3 in the presence (bullet, ) or absence (circle, up triangle, box, down triangle, ) of 1 mM APMA. The incubation was performed at different molar ratios of MMP-3 to proMMP-7; bullet and circle, 0.1 molar; and up triangle, 0.5 molar; and box, 1 molar; and down triangle, 2.5 molar; and , 5 molar. Since the activation curve of proMMP-7 by MMP-3 in the molar ratios of 0.5-5 in the presence of APMA was very similar, it is indicated by . The total activity was measured using [^3H]Cm-Tf substrate for 30 min at 37 °C, and the MMP-7 activity was calculated by subtraction of the activity generated by active MMP-3 in each assay. The full activity of MMP-7 was taken from that of proMMP-7 activated with 1 mM APMA () at 37 °C for 4 h. times shows proMMP-7 incubated with buffer alone. B, conversion of proMMP-7 during incubation with MMP-3. Five molar excess amount of MMP-3 was incubated with proMMP-7 (500 ng) in the absence of APMA at 37 °C for 2 h (lane 2), 4 h (lane 3), 8 h (lane 4), and 24 h (lane 5). Lanes 1 and 6 show proMMP-7 incubated with buffer alone for 0 and 24 h, respectively. The reaction products were subjected to SDS-PAGE (15% total acrylamide) under reducing conditions after termination of the reaction with 20 mM EDTA and the gel was stained with silver nitrate. Arrow and arrowhead indicate active MMP-3 of M(r) 45,000 and 28,000, respectively. The protein bands of MMP-3 are more faintly stained than that of MMP-7 species in the gel.



Activation of ProMMP-1, -2, -3, and -9 by MMP-7

It has been reported that proMMP-1 can be activated by other active MMPs including MMP-3, -7, -10, and -11(3, 12, 13, 36) . Previous studies by us (16) and other groups (15) have also demonstrated that proMMP-9 is activated by MMP-3. Thus, we examined activation of proMMP-1, -2, -3, and -9 by MMP-7. Fig. 5shows that proMMP-1 is activated by MMP-7 in a time- and dose-dependent manner. The activation rate by MMP-7 in a 1:1 molar ratio reached the level obtained by APMA-treatment. When proMMP-1 activation by MMP-7 was performed in the presence of 1 mM APMA, MMP-1 activity was strongly enhanced in a dose-dependent manner: 6.5-fold more activity than that achieved by treatment with MMP-7 or APMA alone was obtained (Fig. 5). There was no difference in the relative molecular masses of active MMP-1 species of M(r) 43,000 generated by treatment of proMMP-1 with APMA and/or MMP-7 (data not shown). However, NH(2)-terminal sequence analyses of these active MMP-1 species showed a different amino acid sequence: APMA-activated MMP-1 had NH(2)-terminal Val-Leu-Thr-Glu-Gly and Leu-Thr-Glu-Gly-Asn in 2:1 molar ratio, whereas the NH(2) terminus of active MMP-1 mediated by MMP-7 in the presence of APMA was Phe-Val-Leu-Thr-Glu-Gly.


Figure 5: Activation of proMMP-1 by MMP-7. ProMMP-1 (137 ng) was incubated with MMP-7 in the presence (bullet, , ) or the absence (circle, up triangle, box) of 1 mM APMA. MMP-1 activity was assayed using [^14C]collagen for 1 h at 37 °C. Since maximal activation of proMMP-1 with 1 mM APMA () was obtained after 4 h of incubation at 37 °C during a period of 0-24 h, the incubation was performed up to 4 h. The molar ratios of MMP-7 to proMMP-1 are 0.1 (bullet, circle), 0.5 (, up triangle), and 1 (, box). Incubation of proMMP-1 with buffer alone (times) shows no activation.



ProMMP-9 was also activated by MMP-7, although the activation rate was confined to 50% of the full activity (Fig. 6A). The activation occurred in a dose-dependent manner, and maximal activation was seen after 4 h of incubation with MMP-7 in a molar ratio of 1:1, the activity of which declined to 20% after 24 h (Fig. 6A). In the presence of both MMP-7 and 1 mM APMA, proMMP-9 was activated to its full activity, and the activation was faster than that by APMA alone (Fig. 6A). Incubation of I-labeled proMMP-9 with MMP-7 in a 1:1 molar ratio converted the zymogen of M(r) 92,000 to a fragment of M(r) 78,000 with intermediate forms with M(r) 83,000 and M(r) 80,000 (Fig. 6B). As previously reported by us(16) , proMMP-9 was processed to a M(r) 67,000 active form with the NH(2)-terminal amino acid sequence of Met-Arg-Thr-Pro-Arg through an intermediate species of M(r) 83,000 during APMA activation. When proMMP-9 activation was performed in the presence of MMP-7 and 1 mM APMA, the active form of M(r) 62,000 was generated via an intermediate species of M(r) 83,000 (data not shown). The M(r) 62,000 species of MMP-9 was associated with 70% of the full activity, whereas only 25% activity was seen with the M(r) 78,000 species. NH(2)-terminal amino acid sequence analyses demonstrated that the M(r) 78,000 species has Leu-Arg-Thr-(Asn)-Leu sequence and the M(r) 62,000 species Met-Arg-Thr-Pro-Arg and Phe-Gln-Thr-Phe-Glu sequence in a 1:1 molar ratio. Immunoblot analyses using monoclonal antibodies specific to NH(2)-terminal (residues 31-49) or the COOH-terminal domain (residues 643-661) of proMMP-9 (16) showed that the M(r) 78,000 species lacks the COOH-terminal domain and the M(r) 62,000 species both NH(2)- and COOH-terminal domains (data not shown).


Figure 6: Time course activation and conversion of proMMP-9 by MMP-7. A, proMMP-9 (215 ng) was incubated with activated MMP-7 in the presence (bullet, , ) or the absence (circle, up triangle, box) of 1 mM APMA. Activity of proMMP-9 incubated with 1 mM APMA () for 24 h at 37 °C was taken as the full activity. Incubation of proMMP-9 with buffer alone (times) shows negligible activation. The incubation was performed in three different molar ratios of MMP-7 to proMMP-9: bullet and circle, 0.1 molar; and up triangle, 0.5 molar; and box, 1 molar. B, a mixture of I-labeled and unlabeled proMMP-9 (860 ng) was incubated with MMP-7 in 1:1 molar ratio at 37 °C for various times as indicated. After termination of the reaction with 20 mM EDTA, the samples were subjected to in SDS-PAGE (9% total acrylamide), and the gel was autoradiographed. Protein standards are as in Fig. 1B. C0, proMMP-9 incubated with buffer alone for 0 h.



In contrast to the effect of MMP-7 on proMMP-1 and proMMP-9, MMP-7 did not activate proMMP-2 even in a 5:1 molar ratio and no definite processing of the proMMP-2 molecule was observed. Treatment of proMMP-3 with MMP-7 resulted in neither activation nor processing of the zymogen (data not shown).

Digestion of Extracellular Matrix Macromolecules

Fibronectin was digested into several smaller fragments with apparent M(r) ranging from 375,000 to 60,000 under non-reducing conditions and fragments with M(r) 190,000, 170,000, and 125,000 remained even after 24 h of incubation. Under reduction, very similar digestion fragments with M(r) 170,000, 155,000, 140,000, 110,000, and 85,000 were seen (data not shown). The digestion patterns were different from those observed by MMP-2 or MMP-3 action(17, 29) . All the alpha1, beta1, and 1 chain of laminin-1 (37) were digested with MMP-7 into fragments with M(r) 140,000 and 110,00 under reducing conditions (data not shown). Type IV collagen was shown to be a substrate of MMP-7: it degraded the alpha2(IV) chain more readily than alpha1(IV) chain, generating a fragment with apparent M(r) of 142,000 at 32 °C. At 35 °C the alpha(IV) chains, especially the alpha2(IV) chain, were digested into small fragments, probably due to partial thermal unfolding of the collagen molecule (data not shown). Type I gelatin was also degraded by MMP-7, and alpha2(I) chains were preferentially digested (data not shown). Although longer incubation caused marked degradation of both alpha2(I) and alpha1(I) chains, gelatinolytic activity of MMP-7 was hardly detected by a solution assay up to 8 h. The prolonged incubation (24 h) gave a specific activity of the enzyme to type I gelatin as 250 µg of gelatin/h/µg of enzyme at 37 °C. MMP-7 did not have the ability to degrade native type I, II, III, V, and VI collagens. Cartilage proteoglycan was digested by MMP-7 with an activity of 400 µg of proteoglycan/h/nmol enzyme at 37 °C. Among MMP-1, -2, -3, -7, and -9, MMP-7 degraded elastin most effectively with a specific activity of 34.2 µg/h/nmol enzyme, whereas MMP-1, -2, -3, and -9 had activities of 0.02, 10.7, 3.1, and 5.8 µg/h/nmol enzyme, respectively.


DISCUSSION

We have purified the precursor of MMP-7 to homogeneity from the culture medium of CaR-1 human rectal carcinoma cells. The NH(2)-terminal sequence analyses of the precursor and APMA-activated MMP-7 and immunoblotting data indicate that the enzyme purified in this study is identical to a zymogen of pump-1, matrilysin (38) .

Like other proMMPs, the enzymic activity was detected only after activation of proMMP-7 with APMA. Our previous studies have demonstrated that the autocatalytic activation of proMMP-2 and proMMP-3 by APMA is concentration dependent and the maximal activation occurs with 1.0 mM APMA for proMMP-2 (17) and 1.5 mM APMA for proMMP-3(18) . Compared with these proMMPs, proMMP-7 is different in that it can be fully activated by a wide range of APMA concentrations, i.e. 0.1-1 mM APMA. Since proMMP-7 has only one cysteine residue in the propeptide region of the molecule and lacks the COOH-terminal hemopexin-like domain, it may be possible that the conformational changes of the molecule by APMA can readily be induced by low concentrations of APMA and become active. During activation with APMA, proMMP-7 of M(r) 28,000 was converted to an active species of M(r) 19,000 with an intermediate form of M(r) 21,000. NH(2)-terminal sequence analysis indicated that cleavage of the Glu-Tyr bond results in the fully active MMP-7 and is identical to the site for activation of recombinant MMP-7(11) . This bond is located at a position three amino acids downstream from the highly conserved Pro-Arg-Cys-Gly-Val/Asn-Pro-Asp sequence of the propeptide of all proMMPs. The hydrolysis of corresponding bonds, the Gln-Phe bond in proMMP-1(13) , the Asn-Tyr bond in proMMP-2 (17, 39) , the His-Phe bond in proMMP-3 (40) , and the Arg-Phe bond in proMMP-9 (15) , is also an essential event for activation of these proMMPs, although proMMP-9 activation by APMA is achieved by sequential processing of both NH(2)-terminal propeptide at a position four amino acids upstream from the conserved sequence and COOH-terminal domain(16) . The step-wise activation, as shown in the present studies, is a common feature of the APMA-mediated activation of proMMPs and has been reported with human recombinant proMMP-7 (promatrilysin)(11) .

Trypsin is an effective activator of proMMP-7 as shown here and previously(11) . However, unlike proMMP-1, -3, and -9, proMMP-7 was not effectively activated with other serine proteinases. Only plasmin and leukocyte elastase activated proMMP-7 up to 50% of the activity. Although cathepsin G had the ability to accelerate the activation processes by plasmin or leukocyte elastase, the activation rate was unchanged. In addition, exposure of proMMP-7 to acid or HOCl, which is known to cause activation of proMMP-8 (41) and proMMP-9(16, 42) , did not show any effects on the activation. These data suggest that serine proteinases and active oxygen metabolites derived from plasma and inflammatory cells may play a limited role in the activation of proMMP-7. Thus, other mechanisms should be necessary for proMMP-7 activation in vivo.

It has been reported that MMP-3 is a good activator of proMMP-1(12, 13) , proMMP-8(14) , and proMMP-9(15, 16) . In the present studies we have demonstrated for the first time that MMP-3 can fully activate proMMP-7 and generate an active species of M(r) 19,000, which has the same NH(2) terminus as that obtained by APMA activation. The activation of proMMP-7 was not associated with formation of intermediate species and was dependent on the concentrations of MMP-3. In addition, this activation process required enzymic activity of MMP-3 because neither proMMP-3 nor MMP-3 inactivated by heat or 1,10-phenanthroline treatment activated the zymogen. Thus, it seems probable that proMMP-7 is directly activated via the cleavage of the Glu-Tyr peptide bond by the action of MMP-3. A similar mechanism has been reported with proMMP-8 activation by MMP-3(14) . Recent studies on tissue localization of MMP-7 have disclosed that MMP-7 is expressed in various carcinoma tissues including stomach, colon, head and neck, lung and prostate carcinoma(43, 44, 45) , glomerular mesangial cells(34) , endometrial gland epithelium(46) , and peripheral blood monocytes(47) . On the other hand, proMMP-3 is also produced by cancer cells(44, 48, 49) , fibroblasts(50) , rheumatoid synovial cells(51) , and endometrial stromal cells (46) and can be stored in the extracellular milieu since both latent and active forms of MMP-3 bind to collagen fibrils(52) . CaR-1 cells used in the present study did not produce MMP-3. However, simultaneous production of MMP-7 and MMP-3 has been demonstrated in human glioma cell lines(49) . Although previous studies showed no coordinated mRNA expression of MMP-7 and MMP-3 in stomach and colon (43) , prostate (45) and breast carcinomas(53) , the co-expression of MMP-7 and stromelysins (MMP-3 and/or MMP-10) has been reported in squamous cell carcinomas of the lung, and head and neck(44) . Actually, our immunolocalization studies on the human colon and lung carcinoma tissues have demonstrated that some carcinoma cells occasionally produce both MMP-7 and MMP-3. (^2)In addition, concomitant expression has been reported in mesangial cells (54) and endometrial tissue(46) . It is well known that proMMP-3 is readily activated by various serine proteinases such as plasmin, leukocyte elastase, cathepsin G, and plasma kallikrein(18) . Thus, these suggest the possibility that MMP-7 participates in the degradation of the extracellular matrix macromolecules in vivo through activation by interaction with MMP-3.

The present studies have demonstrated that MMP-7 can enhance MMP-1 activity 6.5-fold more than that achieved by APMA activation, confirming the previous study using recombinant human MMP-7(8) . This phenomenon was first reported for proMMP-1 activation by MMP-3(12, 55) and then for proMMP-8 activation by MMP-3(14) . In these experiments, the hydrolysis of the specific bonds located three amino acids downstream from the conserved Pro-Arg-Cys-Gly-Val/Asn-Pro-Asp sequence, Gln-Phe for MMP-1 and Gly-Phe for MMP-8, is observed(13, 14) . Although the previous study (8) did not determine the cleavage site of proMMP-1 after activation with MMP-7, our study revealed the cleavage at the same position, i.e. Gln-Phe bond, as that reported with MMP-3 (13) . Thus, it can be concluded that the hydrolysis of the Gln-Phe bond of proMMP-1 is essential for full activation. Recent crystallographic studies on MMP-8 demonstrated that the NH(2)-terminal ammonium group of Phe forms a salt bridge with the side chain carboxylate group of Asp and suggested that this linkage may be related to the full activity of MMP-8 and MMP-1(56) .

We have also demonstrated that MMP-7 can partially activate proMMP-9. Removal of NH(2)-terminal propeptides is believed to be indispensable to formation of active MMP species. However, our previous studies demonstrated that the sequential processing of both NH(2)- and COOH-terminal peptides from the proMMP-9 molecule is essential for full activation, leading to formation of the active species of M(r) 67,000 with the NH(2) terminus of Met-Arg-Thr-Pro-Arg-Cys-Gly-Val-Pro-Asp(16) . On the other hand, O'Connell et al.(57) have recently reported that removal of the COOH-terminal domain is unnecessary for the activation from data using the COOH-terminal-truncated proMMP-9. ProMMP-9 activation by MMP-7 in the present study was associated with removal of both the first NH(2)terminal 15 amino acid residues and COOH-terminal domain. Since Ogata et al.(15) have reported that no enzymic activity is seen with the intermediate species of MMP-9 generated by cleavage at the Glu-Met bond, it seems unlikely that removal of 15 amino acid residues from the NH(2)-terminal propeptide of proMMP-9 is responsible for the activity up to 50%. Thus, proMMP-9 activation by MMP-7 may be achieved through the conformational changes due to the cleavage in both NH(2)- and COOH-terminal peptides of the zymogen. A similar mechanism is proposed for activation of proMMP-11 (prostromelysin 3)(3) . As the activation rate of proMMP-9 by MMP-7 is confined to 50%, the biological function of the activation is unclear. However, it might be possible that proMMP-9 activation is accelerated by interaction with both MMP-3 and MMP-7.

MMP-7 can digest cartilage proteoglycan, type IV collagen, type I gelatin, fibronectin, laminin-1, and insoluble elastin. This substrate specificity of MMP-7 appears to be analogous to that of MMP-3(29) . However, the digestion patterns of type IV collagen, type I gelatin, fibronectin, and laminin-1 by MMP-7 were different from those with MMP-3 of M(r) 28,000 which is a low molecular weight form of MMP-3 lacking both NH(2)- and COOH-terminal domains(18, 29) . In addition, proteoglycan degrading activity of MMP-7 was 1.3-fold higher than that of MMP-3(29) . Among MMP-1, -2, -3, -7, and -9 MMP-7 had the highest activity against insoluble elastin, 11-fold more active than MMP-3. These results indicate that although MMP-7 and MMP-3 share substrates they have different specific activities against each macromolecule. Elastin is the highly cross-linked extracellular matrix component of elastic connective tissues such as blood vessels, lung, and skin. The elastic lamina is present beneath the serosal mesothelial cell lining of the stomach and colon and can function as a barrier to carcinoma cell invasion. Since the enhanced expression of MMP-7 has been reported in stomach and colon cancers(43) , it may be possible that in concert with other MMPs such as MMP-3, MMP-7 produced by the cancer cells plays a part in the breakdown of the elastin as well as other extracellular matrices, which facilitates tumor cell dissemination to the abdominal cavity.


FOOTNOTES

*
This work was supported by Grant-in-aid 05670167 from the ministry of Education, Science and Culture of Japan (to Y. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan. Tel.: 81-762-34-4507; Fax: 81-762-34-4508.

(^1)
The abbreviations used are: MMPs, matrix metalloproteinases; proMMP, corresponding zymogen form; APMA, 4-aminophenylmercuric acetate; DIFP, diisopropyl fluorophosphate; PAGE, polyacrylamide gel electrophoresis; TIMP, tissue inhibitor of metalloproteinases; Cm-Tf, carboxymethylated transferrin.

(^2)
K. Imai, A. Kimura, I. Nakanishi, and Y. Okada, unpublished data.


ACKNOWLEDGEMENTS

We thank R. Tokuda for her skillful technical assistance.


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