Membrane Type 1 Matrix Metalloproteinase Digests Interstitial Collagens and Other Extracellular Matrix Macromolecules*

(Received for publication, June 24, 1996, and in revised form, September 20, 1996)

Eiko Ohuchi Dagger §, Kazushi Imai Dagger , Yutaka Fujii par , Hiroshi Sato **, Motoharu Seiki ** and Yasunori Okada Dagger Dagger Dagger

From the Departments of Dagger  Molecular Immunology and Pathology, and ** Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, § Fuji Chemical Industries, Ltd., Takaoka, Toyama 933, and par  Department of Chemistry, Fukui Medical School, Fukui 910-11, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (Delta MT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha 1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha 2(I) chains. Delta MT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of Delta MT1 and MMP-1 indicate that Delta MT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of Delta MT1 is 8-fold higher than that of MMP-1. Delta MT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha 1-proteinase inhibitor and alpha 2-macroglobulin. The activity of Delta MT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.


INTRODUCTION

Matrix metalloproteinases (MMPs)1 are zinc endopeptidases consisted of 14 different members and implicated in the extracellular matrix (ECM) degradation under both physiological and pathological conditions (1). Among the MMPs, MMP-2 (gelatinase A) is reported to be most related to invasion and metastasis in various human cancers (2). All these MMPs except for at least MMP-11 (stromelysin-3) are secreted as inactive zymogens (proMMPs), and thus their activation is one of the most important steps for the regulation of MMP activities. Research on the activation mechanisms of proMMP-2 has greatly progressed in recent years, since membrane type 1 MMP (MT1-MMP) has been cloned as an activator of proMMP-2 (3). The expression of MT1-MMP in human lung and gastric carcinomas is well correlated with the activation of proMMP-2 (4, 5), suggesting that the proMMP-2 activation by MT1-MMP is a key step for the cancer cell invasion and metastases. On the other hand, one can expect that MT1-MMP possesses enzymic activity to the ECM macromolecules, since the structure of the catalytic domain of MT1-MMP is similar to that of other MMPs. Actually, we have recently demonstrated that a deletion mutant of MT1-MMP lacking the transmembrane domain (Delta MT1) and native MT1-MMP, both of which were isolated in the complex forms with tissue inhibitor of metalloproteinases 2 (TIMP-2), exhibit gelatinolytic activity after separation from TIMP-2 on gelatin zymography (6). However, information about the substrate specificity of MT1-MMP is still limited, although Pei and Weiss (7) have very recently reported that deletion mutants of MT1-MMP have some ECM-degrading activity.

In the present studies, we have purified to homogeneity both Delta MT1 from the stable transfectants and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231) and examined the substrate specificity. The results demonstrate that Delta MT1 and MT1-MMP digest fibrillar collagens, i.e. type I, II, and III collagens, into typical 3/4- and 1/4-length fragments like MMP-1 (tissue collagenase) as well as other ECM components including gelatin, proteoglycan, fibronectin, vitronectin, and laminin-1. In addition, Delta MT1 activates proMMP-2, and the activity on type I collagen is synergistically increased by co-incubation of the substrate with MMP-2.


EXPERIMENTAL PROCEDURES

Materials

Materials were obtained as follows: bovine serum albumin, Brij 35, Coomassie Brilliant Blue R250, 2-mercaptoethanol, transferrin (human), and tosyl-Phe-CH2Cl-trypsin from Sigma; EDTA, iodoacetamide, and sodium dodecyl sulfate from Wako Chem., Japan; p-aminophenylmercuric acetate (APMA) from Aldrich; alpha 1-proteinase inhibitor (human) and alpha 2-macroglobulin (human) from Calbiochem; Dulbecco's modified Eagle's medium, antibiotics, lactalbumin hydrolysate, and fetal calf serum from Life Technologies, Inc.; Green A Dyematrex gel from Amicon Corp. (Beverly, MA); gelatin-Sepharose and Sephadex G-10 from Pharmacia Fine Chemicals, Sweden; DEAE-cellulose (DE-52) from Whatman, UK; Affi-gel 10 from Bio-Rad; [14C]acetic anhydride (20 mCi/mmol), Na125I (16.4 mCi/mg), and [3H]iodoacetic acid (90 mCi/mmol) from Amersham Corp., Japan; a synthetic quenched fluorescent peptide substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, from Peptide Institute, Inc., Japan. ProMMP-1, -2, -3, and -9, and recombinant TIMPs (rTIMP-1 and rTIMP-2) were purified as described previously (8-11). Laminin-1 and type IV collagen were purified from Engelbreth-Holm-Swarm (EHS) tumor (12, 13). Type I collagen from guinea pig and type III, V, and VI collagens from human placenta were isolated as described previously (14, 15). Fibronectin and vitronectin were purified from human plasma (16). 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 methods of Nagase and Woessner (17).

Cell Cultures and Stable Transfectants of Delta MT1

The SV40 early promoter of the pSG5 plasmid (Stratagene, La Jolla, CA) was used to express Delta MT1, which lacks the COOH-terminal transmembrane and cytoplasmic domain of MT1-MMP (Delta Ala536-Val582) (18). A cell line constitutively expressing Delta MT1 was established by two-step selection of CHO cells lacking dihydrofolate reductase gene co-transfected with Delta MT1 cDNA/pSG5 plasmids and pKG5 plasmid containing neomycin resistance gene and dihydrofolate reductase/pSV2 vector as described previously (6). The established cells were cultured in alpha -minimum Eagle's medium containing 0.2% lactalbumin hydrolysate for 4 days. MDA-MB-231 cells were grown in monolayer cultures in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and treated with 20 µg/ml concanavalin A (Wako Chem., Japan) for 4 days in serum-free Dulbecco's modified Eagle's medium containing 0.2% lactalbumin hydrolysate. These culture media were harvested and stored at -20 °C until used for purification.

Purification of Delta MT1 and MT1-MMP from Their TIMP-2 Complex Forms

We have recently isolated Delta MT1 complexed with TIMP-2 from stable transfectants in CHO cells and native MT1-MMP·TIMP-2 complex from concanavalin A-stimulated MDA-MB-231 cells by a four-step protocol (6). The complexes were further subjected to the anti-MMP-1-IgG-Sepharose column to eliminate the possibility of MMP-1 contamination in the preparations, which was monitored by the sandwich enzyme immunoassay and immunoblotting for MMP-1 (19). Delta MT1 and native MT1-MMP were finally purified to homogeneity by application of the complexes to anti-TIMP-2-IgG-Sepharose (clone 67-4H11, the antibody against the COOH-terminal tail domain of TIMP-2) (20) equilibrated with 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM CaCl2, 0.05% Brij 35, and 0.02% NaN3; Delta MT1 and MT1-MMP were separated from TIMP-2 with 5 mM and 10 mM EGTA in the CaCl2-free buffer, respectively, and TIMP-2 was eluted with 6 M urea in the column buffer. Each fraction for the eluate contained 0.5 mM ZnCl2 and 10 mM CaCl2 (at final concentrations) to restore the metal ions immediately after the elution. Delta MT1 and MT1-MMP were monitored by immunoblotting and gelatin zymography as described below. The combined fractions containing Delta MT1 or MT1-MMP were dialyzed against 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35 and 0.02% NaN3. The purified Delta MT1 and MT1-MMP were analyzed on SDS-polyacrylamide gel electrophoresis (PAGE).

Immunoblot Analyses and Gelatinolytic Activity

Samples resolved by SDS-PAGE under reduction were transferred onto nitrocellulose filters. The filters were reacted with a monoclonal antibody against MT1-MMP, and protein bands were visualized by avidin-biotin-peroxidase complex method as described previously (8). The antibody specific to the catalytic domain of MT1-MMP (clone 114-1F2) was prepared and characterized previously (3). For detection of gelatinolytic activity, gelatin zymography and gelatinase assay using heat-denatured 14C-acetylated type I collagen (gelatin) were performed according to the methods described by us (9). Digestion products of the gelatin were also analyzed by SDS-PAGE under reduction (9).

Iodination and Cross-linking Experiments

Delta MT1 and proMMP-2 were iodinated according to the methods by Fraker and Speck (21) and used for the cross-linking experiments and proMMP-2 activation by Delta MT1. For cross-linking experiments, labeled Delta MT1 was incubated with TIMP-2 or proMMP-2 in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35, and 0.02% NaN3 for 2 h at 23 °C, and then the buffer was replaced with 50 mM HEPES-KOH buffer, pH 7.5, 0.15 M NaCl, 5 mM CaCl2, and 0.02% Brij 35 by spin columns (6). Freshly prepared cross-linker (bis(sulfosuccinimidyl) substrate) (Pierce) was added to the samples at a final concentration of 4 mM and incubated for 45 min at 23 °C. After termination of the reactions by incubation with 50 mM Tris for 15 min on ice, they were subjected to SDS-PAGE (10% total acrylamide) under reduction and the gels were autoradiographed. Mr changes of proMMP-2 during activation with Delta MT1 were also examined by autoradiography of the iodinated proMMP-2 after SDS-PAGE (9% total acrylamide).

Determination of Enzyme Concentrations

Concentrations of MMP-1, MMP-2, and Delta MT1 were determined by titration of their activities against rTIMP-2 (concentration determined by amino acid analysis) in an assay using Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 at 37 °C for 1 h (22). Residual enzymic activities were measured and plotted versus TIMP-2 concentrations. A linear plot of activity against the inhibitor molarity was extrapolated to be zero activity at molarity of the enzyme solution.

Digestion of Extracellular Matrix Macromolecules

Delta MT1 was incubated with various ECM components and other protein substrates including carboxymethylated transferrin (23), alpha 1-proteinase inhibitor and alpha 2-macroglobulin in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35, and 0.02% NaN3 at indicated temperatures. The reactions were stopped with 20 mM EDTA and digestion products were analyzed by SDS-PAGE. Specific activity of Delta MT1 and MMP-1 against type I collagen and gelatin was determined using 14C-acetylated type I collagen and its heat-denatured collagen (gelatin), respectively. The degradation of acid-soluble type I (guinea pig), II (bovine), and III (human) collagens was quantitated using the gel scanning protocol described by Welgus et al. (24). The activity of Delta MT1 to cartilage proteoglycan and insoluble elastin were assayed as described previously (17, 25). Synergistic effects of Delta MT1 (or MMP-1) and MMP-2 on type I collagen digestion were assayed using 14C-acetylated collagen.

Sequence Analyses of Delta MT1-digested Type I Collagen

Human type I collagen was digested with Delta MT1 at 27 °C, and the fragments generated were separated by SDS-PAGE, and then transferred on polyvinylidine difluoride membranes. The band of 1/4-length fragments was sequenced by 492 sequencer (Applied Biosystem, Foster, CA).


RESULTS

Purification of Delta MT1 and Native MT1-MMP

Dissociation of Delta MT1 from Delta MT1·TIMP-2 complex was efficiently performed by a stepwise elution method with EGTA and urea in the anti-TIMP-2-IgG-Sepharose column. Delta MT1 was recovered in the EGTA eluate, and the final material (80.3 µg) purified from the culture medium (500 ml) migrated as a single protein band of Mr 56,000 under reducing conditions (Mr 52,000 under nonreducing conditions) (Fig. 1A). Absence of MMP-1 contamination was verified by the sandwich enzyme immunoassay and immunoblotting for MMP-1 (data not shown). Native MT1-MMP was also purified from the MDA-MB-231 cell-derived MT1-MMP·TIMP-2 complex; the final material (5 µg) was purified from 500 ml of the culture medium. Purified MT1-MMP showed a protein band of Mr 56,000 under reducing conditions and that of Mr 52,000 under nonreduction (Fig. 1A). Delta MT1 and native MT1-MMP digested type I gelatin and carboxymethylated transferrin into the indistinguishable fragments (Fig. 1B), indicating that the activities of both enzymes are identical. Both proteinases were already active without any treatment and there was no decrease in the activity even after storage at 4 °C for 4 months. Since a small amount of native MT1-MMP was purified, Delta MT1 was used mainly for the following studies.


Fig. 1. SDS-PAGE of the purified Delta MT1 and native MT1-MMP (A) and of the gelatin digestion products by them (B). A, iodinated Delta MT1 and native MT1-MMP were subjected to SDS-PAGE (10% total acrylamide) under reduction (lanes 1 and 2) and nonreduction (lanes 3 and 4), and the gels autoradiographed. Lanes 1 and 3, Delta MT1; lanes 2 and 4, MT1-MMP. B, human type I gelatin (15 µg) was incubated with purified Delta MT1 (2.8 ng) or native MT1-MMP (2.3 ng) at 37 for 8 h. The digestion products were analyzed by SDS-PAGE (7% total acrylamide) under reduction after termination of the reaction with 20 mM EDTA. Lanes 1 and 4, the gelatin incubated with buffer alone for 0 h and 8 h, respectively; lanes 2 and 3, the substrate digested with Delta MT1 and native MT1-MMP, respectively.
[View Larger Version of this Image (66K GIF file)]


Reconstitution of Delta MT1·TIMP-2 Complex and Activation of ProMMP-2 by Delta MT1

To study the interaction of Delta MT1 with TIMP-2, cross-linking experiments were carried out by incubating iodinated Delta MT1 with TIMP-2. Radioiodination did not cause any significant changes in the intrinsic properties of the proteinase. When the reaction mixture was analyzed by autoradiography after SDS-PAGE, Delta MT1 made a complex with TIMP-2 of ~Mr 73,000, while Delta MT1 alone resulted in a broader band of Mr ~56,000 (Fig. 2). In agreement with our previous data showing that Delta MT1·TIMP-2 complex forms a trimolecular complex with proMMP-2 through the COOH termini of TIMP-2 and proMMP-2 (6), Delta MT1 per se did not make a bimolecular complex with proMMP-2 (Fig. 2). Reconstituted Delta MT1·TIMP-2 complex showed no enzymic activity in the assays using either Mca-peptide or [14C]gelatin, as we have previously reported with the original Delta MT1·TIMP-2 complex (6).


Fig. 2. Cross-linking experiments of the Delta MT1 with proMMP-2 and TIMP-2. Iodinated Delta MT1 (220 ng) was incubated with buffer alone (lanes 1 and 2), proMMP-2 (300 ng, lane 3) or TIMP-2 (84 ng, lane 4). The samples were then reacted with (lanes 2, 3, and 4) or without 4 mM cross-linker (lane 1), and subjected to SDS-PAGE under reduction (10% total acrylamide) as described under "Experimental Procedures." The gel was autoradiographed. Note a band of Delta MT1 complexed with only TIMP-2 (arrowhead).
[View Larger Version of this Image (55K GIF file)]


The action of Delta MT1 on the processing of proMMP-2 was examined. Radiolabeled proMMP-2 was incubated with Delta MT1 in different molar ratios ranging from 1:1 to 1:10, and Mr changes of proMMP-2 molecule were analyzed by autoradiography and gelatin zymography after SDS-PAGE. As shown in Fig. 3A, Delta MT1 processed proMMP-2 of Mr 72,000 into the Mr 69,000 intermediate species in a dose-dependent manner, while APMA treatment generated fully active form of Mr 67,000. On gelatin zymography under nonreduction proMMP-2 of Mr 68,000 was processed by Delta MT1 to the intermediate species of Mr 64,000 and active form of Mr 62,000, the latter of which showed a very faint proteolytic band (Fig. 3B). This processing of proMMP-2 by Delta MT1 confirmed the previous data showing that MT1-MMP initially cleaves proMMP-2 to the intermediate form, which is then autocatalytically processed to the fully active form only when higher concentrations of proMMP-2 are present (26).


Fig. 3. SDS-PAGE (A) and gelatin zymography (B) of proMMP-2 incubated with Delta MT1. A, a mixture of iodinated and unlabeled proMMP-2 (15 ng) was incubated with Delta MT1 in different molar ratios ranging from 1:0 to 1:10 at 37 °C for 24 h, and subjected to SDS-PAGE (9% total acrylamide) under reduction. The gels were autoradiographed. Lane 1, proMMP-2 incubated with buffer alone; lanes 2-5, proMMP-2 incubated with Delta MT1 in 1:1, 1:2.5, 1:5, and 1:10 molar ratios, respectively; lane 6, proMMP-2 incubated with 1 mM APMA at 37 for 45 min. P, proMMP-2; I, intermediate form; A, active form. B, proMMP-2 (15 ng) was incubated with Delta MT1 in the similar conditions as in A and subjected to gelatin zymography under nonreduction. Lanes 1-6 are as in A. Under this condition, the fully active form of MMP-2 generated by Delta MT1 is detected as a faint proteolytic band only in lanes 4 and 5 of B on gelatin zymography.
[View Larger Version of this Image (64K GIF file)]


Degradation of Extracellular Matrix Macromolecules

Digestion of Collagens by Delta MT1 and MT1-MMP

Delta MT1 cleaved type I, II, and III collagens under the nondenaturing conditions, i.e. at 27 °C, generating 3/4- and 1/4-length fragments of these collagens (Fig. 4A). On the other hand, the collagens were degraded into multiple fragments when incubated at 35-37 °C, probably because of thermal denaturation of the substrates to gelatins (data not shown). Since the digestion products were similar to those obtained by the action of MMP-1, NH2-terminal sequence analyses on the 1/4 fragments of type I collagen were performed. The NH2 terminus of alpha 1(I) chains was Ile776-Ala-Gly-Gln-X-Gly-Val-Val-Gly-Leu and alpha 2(I) chains had NH2-terminal Leu776-Leu-Gly-Ala-Hyp-Gly-Ile and Ile782-Leu-Gly-Leu-Hyp-Gly-Ser in approximately 1:2 molar ratio. Native MT1-MMP also digested type I, II, and III collagens into typical 3/4 and 1/4 fragments (Fig. 4B). However, no degradation of type IV, V, and VI collagens by Delta MT1 and MT1-MMP were observed at the nondenaturing temperatures under 33 °C, whereas type IV and V collagens, but not type VI collagen, were digested into fragments at 35 and 37 °C (data not shown).


Fig. 4. Digestion of type I, II, and III collagens with Delta MT1 (A) and native MT1-MMP (B). Type I, II, and III collagens (15 µg each) were incubated with buffer alone (A and B, lanes 1, 3, and 5), with 500 ng of Delta MT1 (A, lanes 2, 4, and 6) or with 100 ng of MT1-MMP (B, lanes 2, 4, and 6) at 27 °C for 24 h. The digestion products were analyzed by SDS-PAGE under reduction after termination of the reaction with 20 mM EDTA. The gels contain 10% total acrylamide except for that for type II collagen digestion with Delta MT1 (A, lanes 3 and 4), which contains 12.5% total acrylamide. Note appearance of characteristic 3/4 fragments of each collagen and 1/4 fragments of type I collagen in the samples incubated with the proteinases. alpha  and beta  chains of each collagen are indicated.
[View Larger Version of this Image (39K GIF file)]


The catalytic efficiency of type I, II, and III collagens by Delta MT1 and MMP-1 was estimated by incubation of the collagens with increasing concentrations of the proteinases. Delta MT1 most preferentially digested type I collagen; the susceptibility of type I collagen was 6.5- and 4-fold higher than that of type II and III collagens, respectively. On the other hand, the activity of MMP-1 to type III collagen was approximately 4.4- and 25.6-fold greater than that to type I and II collagens, respectively. Kinetic analyses of the type I collagen degradation by MMP-1 and Delta MT1 were performed in the samples containing increasing amounts of type I collagen and constant amounts of the proteinases. Lineweaver-Burk plots were constructed from the velocity data, and values of Km and kcat were extracted (Table I). MMP-1 exhibited a Km of 1.3 µM and a kcat of 22.2 molecules of collagen degraded/enzyme molecule/h, whereas a Km of 2.9 µM and a kcat of 7.1 molecules degraded/enzyme molecule/h were obtained with Delta MT1. Thus, the kcat/Km value of Delta MT1 (2.4 µM-1 h-1) for type I collagen was ~7.1-fold less than that of MMP-1 (17.1 µM-1 h-1). Consistent with the data, specific activity of Delta MT1 (6.4 µg/min/nmol) determined by a solution assay using 14C-acetylated type I collagen was approximately 5-fold less than that of MMP-1.

Table I.

Kinetic parameters for Delta MT1 and MMP-1 from the Lineweaver-Burk plots


Km kcat kcat/Km

µM h-1 µM-1h-1
 Delta MT1 2.9 7.1 2.4
MMP-1 1.3 22.2 17.1

Degradation of Other Substrates

Type I gelatin was readily digested into multiple smaller fragments with Delta MT1, and the beta 1, 2(I) chains and alpha 2(I) chains were preferentially degraded compared to alpha 1(I) chains (Fig. 5A). In an assay using 14C-labeled type I gelatin, specific activity of Delta MT1 was 6.3 µg of gelatin degraded/min/nmol of enzyme at 37 °C, which was 8-fold higher than that of MMP-1, while that of MMP-2 was approximately 80-fold higher. Cartilage proteoglycan was also digested by Delta MT1 with an activity of 24.8 µg of proteoglycan degraded/h/nmol enzyme at 37 °C. Delta MT1 degraded fibronectin into five major fragments of Mr 178,000, 144,000, 123,000, 115,000, and 89,000 (under reduction) (Fig. 5B). Vitronectin was also degraded into two fragments with Mr 41,000 and 40,000 (Fig. 5C). The alpha  chain of laminin-1 was slightly hydrolyzed by Delta MT1 (Fig. 5D), but decorin and insoluble elastin were not substrates of Delta MT1. Delta MT1 also processed alpha 1-proteinase inhibitor to a major fragment of Mr 52,500 and alpha 2-macroglobulin to those of Mr 94,000 and 90,000, the digestion products similar to those obtained with MMP-1 (data not shown).


Fig. 5. Degradation of type I gelatin (A), fibronectin (B), vitronectin (C), and laminin-1 (D) with Delta MT1. A, type I gelatin (15 µg) was incubated with Delta MT1 (500 ng) at 37 °C and the reaction products were analyzed on SDS-PAGE (7% total acrylamide) under the reducing conditions. Lane 1, the substrate incubated with buffer alone for 24 h; lanes 2-5, the substrate incubated with Delta MT1 for 2, 4, 8, and 24 h, respectively. B, fibronectin (10 µg) was digested with Delta MT1 (330 ng) at 37 °C for 24 h, and the products were analyzed on SDS-PAGE (6% total acrylamide) under the reducing conditions. Lanes 1 and 2, the substrate incubated with buffer alone and Delta MT1, respectively. C, vitronectin (5.3 µg) was incubated with Delta MT1 (180 ng) at 37 °C for 24 h, and the products were analyzed on SDS-PAGE (12.5% total acrylamide) under the reducing conditions. Lanes 1 and 2 are as in B. D, laminin-1 (15 µg) was incubated with Delta MT1 (180 ng) at 37 °C for 24 h, and the products were subjected to SDS-PAGE (6% total acrylamide) under the reducing conditions. Lanes 1 and 2 are as in B.
[View Larger Version of this Image (57K GIF file)]


Synergistic Effects of Delta MT1 and MMP-2 on Fibrillar Collagen Digestion

To assess the synergistic effect of Delta MT1 and MMP-2 on type I collagen digestion, 14C-labeled type I collagen was incubated at 35 °C with Delta MT1 (16 nM) in the presence of various amounts of MMP-2 ranging from 0 to 16 nM. As shown in Table II, the collagenolytic activity of Delta MT1 was augmented up to 7.3-fold compared with Delta MT1 alone, although MMP-2 itself showed no collagenolytic activity. Similar experiments were performed with MMP-1 (3 nM) and MMP-2 ranging from 0 to 16 nM. The activity of MMP-1 was also 6.1-fold enhanced in the presence of MMP-2. This effect was ascribed to the accelerated degradation of the collagenolytic fragments (gelatin) by MMP-2, since the collagen digestion products generated by Delta MT1 treatment were completely hydrolyzed into peptides in the presence of MMP-2 (Fig. 6A). Similar data was obtained with type II and III collagens incubated with Delta MT1 and MMP-2 (Fig. 6, B and C). On the other hand, no such effect was found with Delta MT1 and MMP-1; the collagenolytic activity in the presence of Delta MT1 and MMP-1 was equal to the sum of both proteinase activities.

Table II.

Synergism of MMP-2 to the degradation of type I collagen by Delta MT1 or MMP-1


MMP-2a Ratio of collagenolytic activity
 Delta MT1 (16 nM) MMP-1 (3 nM)

(nM)
0 1.0 1.0
4 2.5 2.4
8 6.2 5.2
16 7.3 6.1

a  No collagenolytic activity was detected with MMP-2.


Fig. 6. Synergistic digestion of type I, II, and III collagens by Delta MT1 and MMP-2. A, type I collagen (10 µg) was incubated with buffer alone (lane 1), 100 ng of Delta MT1 (lane 2), 100 ng of Delta MT1 and 35 ng of MMP-2 (lane 3), or 35 ng of MMP-2 (lane 4) at 35 °C for 4 h. After termination of the reaction with 20 mM EDTA, the degradation products were analyzed on SDS-PAGE (10% total acrylamide) under the reducing conditions. B and C, type II and III collagens (10 µg each) were incubated with buffer alone (lane 1), 100 ng of Delta MT1 (lane 2), 100 ng of Delta MT1 and 35 ng of MMP-2 (lane 3), or 35 ng of MMP-2 (lane 4) at 35 °C for 4 h, respectively. The digestion products were subjected to SDS-PAGE as described in A. Note that the digestion fragments generated by the action of Delta MT1 are completely hydrolyzed with MMP-2 (lane 3 in A, B, and C).
[View Larger Version of this Image (73K GIF file)]



DISCUSSION

The present studies have demonstrated for the first time that, like interstitial collagenases, i.e. MMP-1 (tissue collagenase), MMP-8 (neutrophil collagenase), and MMP-13 (collagenase-3), both recombinant and native secreted forms of MT1-MMP digest the native fibrillar collagen types I, II, and III into their typical 3/4 and 1/4 fragments. Although MT1-MMP is structurally assigned in the MMP gene family to the MT-MMP subgroup consisting of MT1-, MT2-, MT3-, and MT4-MMPs, the substrate specificity suggests that MT1-MMP is a member of the interstitial collagenases. Delta MT1 and native MT1-MMP were purified from their complex forms with TIMP-2 by dissociation of the complex due probably to the conformational changes caused by EGTA treatment in the anti-TIMP-2-IgG-Sepharose column chromatography. Supplementation of CaCl2 and ZnCl2 to the column fractions restored the structural integrity of the proteinases, since they reconstitute the complex with TIMP-2 and retain stable activities during storage. Although Delta MT1 appears to form a stable complex with TIMP-2 through the binding of the catalytic domain of Delta MT1 with the inhibitor domain of TIMP-2 as demonstrated with the original Delta MT1·TIMP-2 complex (6), the present data do not exclude the possibility that other domains are also involved during the complex formation. Unlike other most MMPs, such as MMP-1, both Delta MT1 and MT1-MMP were secreted into the culture media in active forms. Our recent study (6) suggested that the furin-recognition site, a unique insertion of the RRKR amino acid sequence between the propeptide and catalytic domains of MT1-MMP, is essential to the intracellular processing of Delta MT1. Indeed, Pei and Weiss (7) have very recently demonstrated that furin is responsible for the NH2-terminal processing of MT1-MMP. Since MMP-11, which possesses an insertion containing the RQKR sequence, is also processed to an active form by furin (27), it seems likely that all the members with the R(R/Q)(K/R)R sequences of the MMP gene family, i.e. MT-MMPs and MMP-11, are intracellularly processed to active forms.

Fibrillar collagen types I, II, and III are resistant to many animal proteinases because of their triple helical structures, and they are cleaved only by interstitial collagenases, MMP-1, -8, and -13. CHO cells and MDA-MB-231 cells produced neither MMP-8 nor MMP-13, at least at the protein level determined by immunoblotting.2 Thus, contamination of MMP-1 in the purified preparations was carefully ruled out since it is secreted by both cell lines. During the purification steps, proMMP-1 was eliminated by using anti-MMP-1-IgG-Sepharose and anti-TIMP-2-IgG-Sepharose column chromatographies, and no contamination in the preparations was verified by sandwich enzyme immunoassay and immunoblotting for MMP-1. This was also supported by the data that the final products have a single protein band of Mr 56,000 and were already active without any treatment for activation. In addition, the collagenolytic activity of Delta MT1 was different from that of MMP-1. Delta MT1 preferentially cleaved type I collagen over type II and III collagens, whereas MMP-1 cleaved preferentially type III collagen over type I and II collagens. Comparison of the type I collagenolytic activity of Delta MT1 with that of MMP-1 revealed that Delta MT1 is 5-~7.1-fold less efficient than MMP-1. In contrast, gelatinolytic activity of Delta MT1 was 8-fold higher than MMP-1. Knäuper et al. (28) recently reported that three collagenases have distinct substrate specificity to collagens and gelatins; MMP-1, -8, and -13 preferentially digest collagen types III, I, and II, respectively, and both MMP-8 and MMP-13 exhibit 4.9- and 41-fold higher activity against type I gelatin than does MMP-1. Thus, the present data suggest that Delta MT1 shares the proteolytic characteristics with MMP-8. Higher gelatinolytic efficiency of MMP-8, MMP-13, and gelatinases (MMP-2 and -9) is explained by the presence of key residues specifically conserved in the active sites of these MMPs (28). Delta MT1 also conserves the residues including Ile in the S'1-pocket, negatively charged Glu just preceding the third His residue and invariant Pro three amino acids after the His residue.

It has been established that MMP-1 and MMP-8 cleave type I, II, and III collagens at a specific single site after the Gly residue of the partial sequences Gly-(Ile or Leu)-(Ala or Leu) located approximately 3/4 from the NH2 terminus in these collagens. Unlike MMP-1 and MMP-8, however, MMP-13 hydrolyzes alpha  chains of type II collagen at the Gly906-Leu907 and Gly909-Gln910 bonds. The present study also demonstrated that Delta MT1 cleaves the Gly775-Leu776 and Gly781-Ile782 bonds of alpha 2(I) chains. Since alpha 1(I) chains were hydrolyzed only at the Gly775-Leu776 bond, cleavage of the Gly781-Ile782 bond may be a secondary cleavage. This two-site cleavage by Delta MT1 may be related with higher gelatinolytic activity of this enzyme, but its biological function remains unclear at the present time. Pei and Weiss (7) have reported that a deletion mutant of MT1-MMP (Delta Pro509-Val582) (MT1-MMP1-508) digests several ECM macromolecules including gelatin, fibronectin, laminin, vitronectin, and dermatan sulfate proteoglycan. However, MT1-MMP1-508 had no ability to cleave type I collagen. The structural difference between Delta MT1 in the present study and MT1-MMP1-508 is that Delta MT1 is longer with 27 amino acid residues in its COOH terminus. Since the substrate specificity of Delta MT1 is almost identical to that of MT1-MMP1-508 except for the activity to fibrillar collagens, it seems likely that the COOH-terminal sequence of the 27 amino acid residues is essential to the collagenolytic activity probably because the sequence is necessary for the intact conformation of the hemopexin-like domain of MT1-MMP, which may interact with the collagen molecules. The present data that MT1-MMP derived from MDA-MB-231 cells also possesses collagenolytic activity indicate that collagenase activity of MT1-MMP is not artificial, but natural.

Our previous study (6) demonstrated that active Delta MT1 has Ala113 at the NH2 terminus, indicating that the Tyr112 is lost during the intracellular activation. In MMP-1 and MMP-8, the Phe at their NH2 termini, which corresponds to Tyr112 of Delta MT1, is essential to keep the conformational integrity and express their full activity. Lack of the Phe in these species generated by APMA activation results in only partial collagenase activity of MMP-1 and MMP-8 (29, 30). Thus, it may be possible that the Tyr112-Delta MT1 would have higher activity against the fibrillar collagens than the Ala113-Delta MT1. When Delta MT1 purified from cDNA-transfected Escherichia coli was treated with furin, Tyr112-Delta MT1 was obtained by the cleavage at the Arg111-Tyr112 bond.3 Comparative study on the collagenolytic activities of Tyr112-Delta MT1 and Ala113-Delta MT1 is under way in our laboratory.

The degradation of the fibrillar collagens is considered to be sequentially performed by gelatinases after the initial cleavage at the collagen triple helix by collagenases (1). A synergistic effect of MMP-1 and MMP-2 on the fibrillar collagen digestion has been reported (31) and further confirmed in the present study. Similar accelerated digestion of the collagens was demonstrated with Delta MT1 and MMP-2. The combination of MT1-MMP and MMP-2 may be crucial for the pericellular collagen degradation in cancer invasion and metastasis, because MT1-MMP can activate proMMP-2 on the carcinoma cell surfaces where both MMPs may act in concert. Indeed, we have demonstrated that MMP-2 is localized on the cell membranes of the MT1-MMP expressing carcinoma cells in human stomach cancers (5). It is also notable that Delta MT1 digests aggrecan, even if the specific activity in vitro is approximately one-third of MMP-3 activity. The initial event in osteoarthritic cartilage is depletion of aggrecan from the articular cartilage, leading to loss of tensile strength of the tissue. So-called "aggrecanase" which clips the aggrecan molecules at the Glu373-Ala374 bond is reported to be a key enzyme for the cartilage degradation (32). Although aggrecanase is thought to be a metalloproteinase (33), none of MMPs except for MMP-8 cleaves the Glu373-Ala374 bond. MMP-8 can digest aggrecan molecules at the aggrecanase-site only when the enzyme is incubated with the substrate in a very high concentration (34). Previous studies (35) have demonstrated that the aggrecanase activity is a cell-mediated event, suggesting the pericellular proteolysis of aggrecan. In fact, our preliminary studies showed that MT1-MMP is highly co-expressed with MMP-2 in human osteoarthritic chondrocytes.4 It is, therefore, reasonable to speculate that aggrecanase may be MT1-MMP or combined action of MT1-MMP and MMP-2. This possibility should be elucidated by further studies.


FOOTNOTES

*   This work was supported by Grant-in-Aid 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Recipient of research fellowships from the Japan Society for the Promotion of Science for Young Scientists.
Dagger Dagger    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; E-mail: yasokada{at}kenroku.ipc.kanazawa-u.ac.jp.
1    The abbreviations used are: MMP, matrix metalloproteinase; proMMP, corresponding zymogen form; MT-MMP, membrane-type MMP; Delta MT1, MT1-MMP lacking transmembrane domain; APMA, p-aminophenylmercuric acetate; PAGE, polyacrylamide gel electrophoresis; TIMP-2, tissue inhibitor of metalloproteinases 2; ECM, extracellular matrix; CHO, Chinese hamster ovary; Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl.
2    E. Ohuchi K. Imai, and Y. Okada, unpublished data.
3    H. Sato and M. Seiki, manuscript in preparation.
4    K. Imai, S. Ohta, and Y. Okada, unpublished data.

Acknowledgments

We are grateful to Dr. K. Iwata (Fuji Chemical Industries, Ltd.) for providing us with monoclonal antibodies. We also thank S. Makino and M. Takegami for their skillful technical assistance.


REFERENCES

  1. Nagase, H., and Okada, Y. (1997) in Textbook of Rheumatology (Kelley, W. N., Harris, E. D., Jr., Ruddy, S., and Sledge, C. B., eds), W. B. Saunders, Philadelphia in press
  2. Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L. A. (1993) Annu. Rev. Cell Biol. 9, 541-573 [CrossRef]
  3. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65 [CrossRef][Medline] [Order article via Infotrieve]
  4. Tokuraku, M., Sato, H., Murakami, S., Okada, Y., Watanabe, Y., and Seiki, M. (1995) Int. J. Cancer 64, 355-359 [Medline] [Order article via Infotrieve]
  5. Nomura, H., Sato, H., Seiki, M., Mai, M., and Okada, Y. (1995) Cancer Res. 55, 3263-3266 [Abstract]
  6. Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710 [Abstract]
  7. Pei, D., and Weiss, S. J. (1996) J. Biol. Chem. 271, 9135-9140 [Abstract/Free Full Text]
  8. Okada, Y., Gonoji, Y., Naka, K., Tomita, K., Nakanishi, I., Iwata, K., Yamashita, K., and Hayakawa, T. (1992) J. Biol. Chem. 267, 21712-21719 [Abstract/Free Full Text]
  9. Okada, Y., Morodomi, T., Enghild, J. J., Suzuki, K., Yasui, A., Nakanishi, I., Salvesen, G., and Nagase, H. (1990) Eur. J. Biochem. 194, 721-730 [Abstract]
  10. Okada, Y., Harris, E. D., Jr., and Nagase, H. (1988) Biochem. J. 254, 731-741 [Medline] [Order article via Infotrieve]
  11. Hayakawa, T., Yamashita, K., Ohuchi, E., and Shinagawa, A. (1994) J. Cell Sci. 107, 2373-2379 [Abstract/Free Full Text]
  12. Kleinman, H. K., McGarvey, M. L., Liotta, L. A., Robey, P. G., Tryggvason, K., and Matrin, G. R. (1982) Biochemistry 21, 6188-6193 [Medline] [Order article via Infotrieve]
  13. Yurchenco, P. D., and Furthmayr, H. (1984) Biochemistry 23, 1839-1850 [Medline] [Order article via Infotrieve]
  14. Minamoto, T., Ooi, A., Okada, Y., Mai, M., Nagai, Y., and Nakanishi, I. (1988) Hum. Pathol. 19, 815-821 [Medline] [Order article via Infotrieve]
  15. Odermatt, E., Risteli, J., van Delden, V., and Timpl, R. (1983) Biochem. J. 211, 295-302 [Medline] [Order article via Infotrieve]
  16. Ruoslahti, E., and Engvall, E. (1978) Ann. N. Y. Acad. Sci. 312, 178-191 [Medline] [Order article via Infotrieve]
  17. Nagase, H., and Woessner, J. F., Jr. (1980) Anal. Biochem. 107, 385-392 [Medline] [Order article via Infotrieve]
  18. Cao, J., Sato, H., Takino, T., and Seiki, M. (1995) J. Biol. Chem. 270, 801-805 [Abstract/Free Full Text]
  19. Zhang, J., Fujimoto, N., Iwata, K., Sakai, T., Okada, Y., and Hayakawa, T. (1993) Clin. Chim. Acta 219, 1-14 [CrossRef][Medline] [Order article via Infotrieve]
  20. Fujimoto, N., Zhang, J., Iwata, K., Shinya, T., Okada, Y., and Hayakawa, T. (1993) Clin. Chim. Acta 220, 31-45 [Medline] [Order article via Infotrieve]
  21. Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80, 849-857 [Medline] [Order article via Infotrieve]
  22. Knight, C. G., Willenbrock, F., and Murphy, G. (1992) FEBS Lett. 296, 263-266 [CrossRef][Medline] [Order article via Infotrieve]
  23. Okada, Y., Nagase, H., and Harris, E. D., Jr. (1986) J. Biol. Chem. 261, 14245-14255 [Abstract/Free Full Text]
  24. Welgus, H. G., Jeffrey, J. J., and Eisen, A. Z. (1981) J. Biol. Chem. 256, 9511-9515 [Free Full Text]
  25. Banda, M. J., and Werb, Z. (1987) Methods Enzymol. 144, 288-305 [Medline] [Order article via Infotrieve]
  26. Sato, H., Takino, T., Kinoshita, T., Imai, K., Okada, Y., Stetler-Stevenson, W. G., and Seiki, M. (1996) FEBS Lett. 385, 238-240 [CrossRef][Medline] [Order article via Infotrieve]
  27. Pei, D., and Weiss, S. J. (1995) Nature 375, 244-247 [CrossRef][Medline] [Order article via Infotrieve]
  28. Knäuper, V., López-Otín, C., Smith, B., Knight, G., and Murphy, G. (1996) J. Biol. Chem. 271, 1544-1550 [Abstract/Free Full Text]
  29. Suzuki, K., Enghild, J. J., Morodomi, T., Salvesen, G., and Nagase, H. (1990) Biochemistry 29, 10261-10270 [Medline] [Order article via Infotrieve]
  30. Knäuper, V., Wilhelm, S. M., Seperack, P. K., DeClerck, Y. A., Langley, K. E., Osthues, A., and Tschesche, H. (1993) Biochem. J. 295, 581-586 [Medline] [Order article via Infotrieve]
  31. Murphy, G., Gavrilovic, J., and McAlpine, C. (1986) in Proteinases in Inflammation and Tumor Invasion (Tschesche, H., ed), pp. 173-187, Walter de Gruyter & Co., Berlin
  32. Sandy, J. D., Flannery, C. R., Neame, P. J., and Lohmander, L. S. (1992) J. Clin. Invest. 89, 1512-1516 [Medline] [Order article via Infotrieve]
  33. Buttle, D. J., Handley, C. J., Ilic, M., Saklatvala, J., Murata, M., and Barrett, A. J. (1993) Arthritis Rheum. 36, 1709-1717 [Medline] [Order article via Infotrieve]
  34. Fosang, A. J., Last, K., Neame, P. J., Murphy, G., Knäuper, V., Tschesche, H., Hughes, C. E., Caterson, B., and Hardingham, T. E. (1994) Biochem. J. 304, 347-351 [Medline] [Order article via Infotrieve]
  35. Lark, M. W., Gordy, J. T., Weidner, J. R., Ayala, J., Kimura, J. H., Williams, H. R., Mumford, R. A., Flannery, C. R., Carlson, S. S., Iwata, M., and Sandy, J. D. (1995) J. Biol. Chem. 270, 2550-2556 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.