©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism Of Cell Surface Activation Of 72-kDa Type IV Collagenase
ISOLATION OF THE ACTIVATED FORM OF THE MEMBRANE METALLOPROTEASE (*)

(Received for publication, September 14, 1994; and in revised form, November 28, 1994)

Alex Y. Strongin (§) Ivan Collier Gregory Bannikov Barry L. Marmer Gregory A. Grant (1) Gregory I. Goldberg

From the Division of Dermatology Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110-1093

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Matrix metalloproteases are secreted by mammalian cells as zymogens and, upon activation, initiate tissue remodeling by proteolytic degradation of collagens and proteoglycans. Activation of the secreted proenzymes and interaction with their specific inhibitors determine the net enzymatic activity in the extracellular space. We have previously demonstrated that 72T4Cl can be activated by a plasma membrane-dependent mechanism specific for this enzyme. Here, we report purification of the membrane activator of 72T4Cl, which is a new metalloprotease identical to a recently cloned membrane-type matrix metalloprotease (MT-MMP). We demonstrate that activated MT-MMP acts as a cell surface tissue inhibitor of metalloprotease 2 (TIMP-2) receptor with K = 2.54 times 10M. The activatorbulletTlMP-2 complex in turn acts as a receptor for 72T4Cl (K = 0.56 times 10M), binding to the carboxyl-end domain of the enzyme. Activation of 72T4Cl on the cell membrane provides a basic mechanism for spatially regulated extracellular proteolysis and presents a new target for prognosis and treatment of metastatic disease. The activator, purified as a tri-molecular complex of MT-MMPbulletTIMP2bulletcarboxyl-end domain of 72T4Cl, is itself an activated form of MT-MMP, posing the following question: what is the mechanism of the activator's activation?


INTRODUCTION

Normal physiological processes such as morphogenesis, tissue repair, angiogenesis, uterine involution, and bone resorption are dependent upon spatial and temporal regulation of extracellular proteolysis. Metalloproteases secreted by eucaryotic cells can initiate tissue remodeling by degradation of ECM (^1)macromolecules such as collagen and proteoglycans(1, 2, 3) . Malignant cells can exploit these same proteases to promote invasion and metastasis(4, 5, 6) . All enzymes of this group, identified so far, are secreted in a soluble proenzyme form. Proenzyme activation(7, 8, 9, 10, 11, 12, 13, 14) and interaction with the specific inhibitors TIMP-1 (14, 15, 16, 17, 18) and TIMP-2 (19, 20, 21) determine the net activity of secreted enzymes. The understanding of the mechanisms determining the fate of metalloproteases after secretion and providing for a spatial regulation of their activity has remained an elusive goal.

We (22) and others (23, 24, 25, 26, 27) have demonstrated a ``plasma membrane-dependent'' mechanism, specific for activation of 72T4Cl, that can be induced in normal and tumorigenic human cells by treatment with TPA or concanavalin A. Activation of 72T4Cl in this system results in the cleavage of the amino-terminal propeptide generating a 64-kDa conversion intermediate with an amino terminus at Leu, which in turn is converted to a Tyr 62-kDa active form (22) . TIMP-2 and a 26-kDa peptide derived from the carboxyl domain of the 72T4Cl competitively inhibit membrane-dependent enzyme activation (22, 23) . Here, we report the isolation of an active form of a membrane-bound activator of 72T4Cl, which is a new membrane-associated metalloprotease, MT-MMP. The activator acts as a cell surface TIMP-2 receptor. The resulting activatorbulletTIMP-2 complex serves as a receptor for the carboxyl-end domain of 72T4Cl, the binding of which causes enzyme activation. While this report was in preparation, Sato et al.(28) cloned MT-MMP, using degenerate polymerase chain reaction, and demonstrated that it causes activation of 72T4Cl upon transfection into Cos-1 cells.


MATERIALS AND METHODS

Cell Culture

HT1080 fibrosarcoma and p2AHT2a (29) cells were grown in monolayer culture in RPMI 1640 media supplemented with 4% fetal calf serum and 2 mM glutamine in the presence of 5% CO(2) and treated with TPA (50 ng/ml for 16-24 h).

Purification of 72T4Cl

The 72T4Cl procollagenase was purified from conditioned medium of the transfected cell line p2AHT7211A as described(22) .

Expression and Purification of TIMP-2

The p6Rhyg expression plasmid was constructed as described earlier(13) . The complete TIMP-2 cDNA was isolated from a skin fibroblast cDNA library (30) and subcloned into the HindIII site of the p6Rhyg vector. The resulting expression plasmid p6RTIMP-2hyg was transfected into E1A-expressing p2AHT2a cells (29) using the calcium-phosphate method (31) as described for 92T4Cl(13) . The stably transfected cell line p2AHTTIMP-2 expressing TIMP-2 was selected in the presence of hygromycin (200 µg/ml). The TIMP-2 was purified from serum-free conditioned media of transfected cell line p2AHTTIMP-2. Media, containing 10 mM Tris, pH 7.5, was applied onto a 200-ml bed volume column of Reactive Red-120-agarose (Sigma, R-0503) equilibrated with 20 mM Tris buffer, pH 7.5, 5 mM CaCl(2) (buffer A) containing 150 mM NaCl. The column was eluted with a linear gradient of 150 mM to 2.5 M NaCl in buffer A. Fractions containing TIMP-2 were revealed by Western blotting with anti-TIMP-2 antibodies, dialyzed against 20 mM Tris (pH 8.6) buffer containing 10 mM NaCl, and applied onto a Q-Sepharose fast flow column (Pharmacia Biotech Inc., 17-0510-01) equilibrated with with the same buffer. The Q-Sepharose column was eluted with a linear gradient of 10-600 mM NaCl in 20 mM Tris, pH 8.6. A well separated peak of TIMP-2 was eluted at 200 mM NaCl. TIMP-2 fractions were pooled and dialyzed against 20 mM NaPO(4) (pH 6.3) buffer containing 10 mM NaCl and applied onto a CM-Sepharose CL-6B (Sigma, CCL-6B-100) column equilibrated with the same buffer. The CM-Sepharose column was eluted with a linear gradient of 10 mM to 500 mM NaCl. TIMP-2 fractions were analyzed by SDS-PAGE and applied onto reverse phase high pressure liquid chromatography if further purification was needed.

Expression and Purification of the FLAG-CT Fusion Protein

Expression vector pFLAG72CT was constructed by cloning a fragment from 72T4Cl cDNA (32) coding for Leu-Cys into Escherichia coli secretion vector pFlag1 (IBI Inc.). The resulting vector coding for the fusion protein FLAG-CT was transfected into an E. coli TOPP5 host (Stratagene). The cells were grown in LB media at 30 °C and induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 1 h. The cells were pelleted, and periplasmic proteins were extracted by osmotic shock essentially as described(33) . The resulting protein solution was clarified by centrifugation (40,000 rpm for 1 h) and applied onto a Reactive Red-120-agarose column equilibrated with buffer A. The column was eluted with buffer A containing 2 M NaCl, and the sample was dialyzed against buffer A containing 150 mM NaCl. The dialyzed protein was applied on anti-FLAG-M1 monoclonal antibody column equilibrated with the same buffer, eluted with 2 mM EDTA, and dialyzed against 25 mM HEPES-KOH buffer, pH 7.5, containing 0.1 mM CaCl(2).

Anti-TIMP-2 rabbit antibodies (a generous gift from Dr. H. Birkedal-Hansen) were affinity purified on immobilized TIMP-2 as described(34) . After extensive washing of unbound proteins, affinity-purified antibodies were eluted with 0.2 M glycine-HCl buffer, dialyzed against phosphate-buffered saline, and stored at -80 °C.

The cleavage of 72T4Cl, isolation of the carboxyl-end domain fragment, reduction-alkylation, and CNBr cleavage were performed as described (22) .

Cross-linking Experiments

Isolation of cell plasma membranes and extraction with Lubrol were performed as described (22) . Ligands were labeled with I using Iodogen (Pierce) according to the manufacturer's instructions. Unincorporated I was removed on a Sephadex G-25 Quick Spin column (Boehringer Mannheim) equilibrated with 0.025 M HEPES buffer, pH 7.5, containing 150 mM NaCl and 0.001% Brij 35. Cross-linking was performed in 10 µl of total volume containing indicated amounts of radiolabeled ligand, competing ligand, plasma membrane, or Lubrol extract protein in 25 mM HEPES buffer, pH 7.5, containing 150 mM KCl. The reaction was incubated for 15 min at 37 °C prior to addition of bis(sulfosuccinimidyl) suberate (BS^3, Pierce) to reach a final concentration as indicated. The reaction was incubated for 1 h at the indicated temperature and stopped by addition of 5 µl of SDS-PAGE sample buffer and 1.5 µl of 100 mM dithiothreitol. Samples were heated for 2 min in a boiling water bath prior to loading on 7.5% SDS-PAGE. The gels were subjected to autoradiography.

Affinity Chromatography of Plasma Membrane Extract from HT1080 Cells

1 mg of purified FLAG-CT fusion protein was loaded onto a column of anti-FLAG monoclonal M1 antibody resin (IBI), equilibrated with buffer A (10 mM HEPES buffer, pH 7.5, containing 150 mM KCl, 1 mM Ca, and 0.002% Brij 35). The column was washed with buffer A and then with buffer A containing 1% Lubrol. A Lubrol extract of plasma membranes from HT1080 cells (100 mg of protein) was applied onto the column, washed with buffer A, and then with buffer A containing 0.1% Lubrol. The bound proteins were eluted with buffer A without Ca containing 2 mM EDTA. Ten 500-µl fractions were collected and analyzed by gel electrophoresis. The fractions of interest were electroblotted on polyvinylidene difluoride membrane, and proteins were sequenced as described.

Zymogram analysis, Western blot, gel electrophoresis, and plasma membrane activation of 72T4Cl were performed as described(22) .

Cell Surface Binding of Radiolabeled Ligands

The cells were grown as above in 12-well clusters, washed with serum-free media containing 25 mM HEPES buffer, pH 7.3, and 1 mg/ml of bovine serum albumin, and incubated on ice for 20 min with (background) or without unlabeled purified TIMP-2 or FLAG-CT fusion ligand prior to addition of I-radiolabeled ligand to reach indicated concentrations. The cells were incubated for 40 min on ice, washed five times with serum-free media, and lysed in 0.4 M NaOH, 0.3% SDS; the radioactivity was then counted. One well of each cluster was used to count the number of cells. The nonspecific background binding was determined using 500-fold excess of unlabeled over highest concentration of I ligand. Each experimental point was measured in duplicate.


RESULTS

Cell Surface Activation of 72T4Cl Is Mediated by a Tri-molecular Complex between Membrane-associated Activator, TIMP-2, and Carboxyl-end Domain of the Enzyme

We have previously shown (22) that TPA-induced HT1080 fibrosarcoma cells contain a specific membrane-associated activator of the 72T4Cl proenzyme. The membrane-dependent activation of exogenously added purified 72T4Cl results in amino-terminal cleavage of the proenzyme, which is blocked by an excess of TIMP-2. The inhibitor, TIMP-2, forms a specific non-covalent stoichiometric complex with 72T4Cl (20) through interaction with the carboxyl-end domain of the proenzyme(35) . This complex is resistant to activation by plasma membranes(22) . In addition, the 26-kDa peptide derived from the carboxyl-end domain of the proenzyme that interacts with TIMP-2 is also able to competitively inhibit membrane-dependent activation(22) . Accordingly, the 72T4Cl truncated at the carboxyl end is resistant to activation by membranes isolated from the fibroblasts stimulated with concanavalin A(25) . These results indicate that the interaction between the carboxyl-end domain of the enzyme and the membrane-associated component is essential for membrane-dependent activation of the enzyme. Thus, we expressed and purified the 26-kDa Leu-Cys carboxyl-end domain fragment of 72T4Cl (32) in the form of an amino-terminal fusion with the tag hexapeptide FLAG. The expression was optimized using an E. coli expression vector pFLAG-1 (IBI) that allows secretion of the recombinant product into the periplasm. Complete purification of the carboxyl-end domain peptide from a periplasm fraction was achieved using red affinity-agarose and monoclonal anti-Flag-M1 antibody columns as described under ``Materials and Methods.''

The inhibitory activity of the carboxyl-end peptide is dependent on its native conformation(22) . Reduction of the disulfide bridge Cys-Cys or partial cleavage with CNBr led to a loss of its inhibitory activity. The 26-kDa fragment was shown to interact with purified TIMP-2 or to form a dimer(22) . Formation of the complex with the inhibitor and dimerization of the fragment are mutually exclusive. This is demonstrated by the fact that an excess of the 26-kDa fragment is able to compete the formation of the complex with TIMP-2 with concomitant appearance of the 52-kDa carboxyl-end domain dimer(22) , and the formation of this dimer is abolished in the presence of a molar excess of TIMP-2. These observations provide a sensitive way to establish whether native and recombinantly expressed fragments of 72T4Cl have a similar conformation and activity. Purified recombinant FLAG-CT was assayed for (i) the presence of a disulfide bridge between 2 Cys residues in the domain; (ii) its ability to inhibit membrane-dependent activation of 72T4Cl; and (iii) its ability to bind TIMP-2. All three parameters were similar or identical to those of native carboxyl-terminal domain peptide obtained from the 72T4Cl enzyme. SDS-PAGE analysis of the purified FLAG-CT fusion under reducing and non-reducing conditions demonstrates the shift in migration characteristic of the presence of the disulfide bridge (Fig. 1A). Competitive inhibition of membrane activation of 72T4Cl and binding of TIMP-2 by FLAG-CT protein was as efficient as the carboxyl-end domain fragment derived from the enzyme (Fig. 1, B and C). Increasing the concentration of I-labeled 26-kDa CT fragment leads to the formation of a 52-kDa dimer (Fig. 1C, lanes1 and 2), as previously described(22) . Based on these results, we used the FLAG-CT fusion protein in place of the proenzyme-derived peptide for cross-linking experiments, as well as for affinity chromatography of the HT1080 plasma membrane extract (see below).


Figure 1: Characterization of the FLAG-CT fusion protein. A, the FLAG-CT fusion protein was purified from periplasm extract of E. coli TOPP5 host (Stratagene) on red and anti-FLAG-M1 monoclonal antibody columns, as described under ``Materials and Methods,'' and analyzed by SDS-PAGE either before (A, lane1) or after (A, lane2) reduction in 10 mM dithiothreitol. B, 72T4Cl (lane4, 15 ng) was activated with plasma membranes (20 µg) from HT1080 cells (lane1) as described under ``Materials and Methods'' with the addition of 400 ng of either 72T4Cl-derived 26-kDa CT (lane2) or recombinant FLAG-CT fusion protein (lane3) prior to the addition of 72T4Cl. C, either I-labeled 26-kDa CT (200,000 cpm, 10^7 cpm/µg, lanes1-4) or I-labeled FLAG-CT fusion (160,000 cpm, 0.5 times 10^7 cpm/µg, lane5) were cross-linked with BS^3 (2 mM) for 1 h at 0 °C in 25 mM HEPES buffer, pH 7.5, containing 150 mM KCl in the presence of 100 ng of unlabeled 26-kDa CT (lane1), FLAG-CT (lane2), FLAG-CT and TIMP-2 (lane3), 26-kDa CT and TIMP-2 (lane4), or TIMP-2 (lane5). The numbers show molecular masses of the markers (A), 72T4Cl and conversion intermediates (B), and 26-kDa CT dimer and complex with TIMP-2 (C).



Cross-linking experiments using I-labeled 26-kDa CT and HT1080 plasma membranes or their Lubrol extract demonstrate the presence of two specific products with apparent molecular masses of 105 and 44 kDa (Fig. 2). The formation of both cross-linking products is competitively inhibited in the presence of the excess of unlabeled 26-kDa CT (Fig. 2, lane4). Reduction-alkylation of the 26-kDa CT abolished its inhibitory activity in the cross-link assay (not shown). An identical result was obtained after the 26-kDa CT was cleaved with CNBr (Fig. 2, lane5). Both the internal fragment (not shown), produced by cleavage at Met and Met, and a fragment containing a single cleavage at Met alone lacked the ability to inhibit the formation of the specific cross-linking products. These findings demonstrate that modifications of the 26-kDa CT that abolish its ability to interact with TIMP-2 also abolish its inhibitory activity (22) in the 72T4Cl membrane activation assay and its ability to interact with the membrane components.


Figure 2: Cross-linking of radiolabeled 26-kDa CT to plasma membrane extracts from TPA-treated HT1080 and p2AHT2a cells. The I-labeled 26-kDa CT (1.2 times 10^6 cpm, 10^7 cpm/µg, lanes1-7 or 1.5 times 10^5 cpm, 0.8 times 10^7 cpm/µg, lanes8-10) in 25 mM HEPES-KOH buffer, pH 7.5, containing 150 mM KCl and 0.1 mM CaCl(2) were incubated with lubrol (1%) extracts of plasma membranes from HT1080 cells (30 µg of protein, lanes2-5) or from p2AHT2a cells (20 µg, lane8, or 40 µg, lanes9 and 10) with (lanes1 and 3-10) or without 2 mM of BS^3 (lane2) for 1 h at 0 °C. Unlabeled ligands, 26-kDa CT (300 ng, lanes4, 7, and 10), or CNBr-cleaved 26-kDa CT (400 ng, lanes5 and 6) were added to the reaction for competition. After incubation, the reactions were analyzed by SDS-PAGE under reducing conditions followed by autoradiography. The numbers show molecular masses of the specific cross-linked products.



The molecular mass of the 44-kDa cross-linking product is indistinguishable from that of the 26-kDa CT-TIMP-2 cross-link(22) . The p2AHT2a cells, expressing the adenovirus E1A gene, despite transcribing TIMP-2 mRNA, do not secrete appreciable amounts of TIMP-2. (^2)Thus, to ascertain whether membrane associated TIMP-2 may be an essential element in membrane-dependent activation, we investigated the ability of the plasma membrane preparations from p2AHT2a cells (29) to yield the 44-kDa cross-linking product with FLAG-CT and activate 72T4Cl proenzyme. Cross-linking experiments using plasma membranes prepared from these cells (Fig. 2, lanes 8-10) demonstrate no specific cross-link products except the 52-kDa dimer of I-labeled 26-kDa CT (Fig. 2, lanes 8-10), which is indistinguishable from the dimer found in the presence of the 26-kDa CT ligand alone (Fig. 2, lane7).

The results presented in Fig. 3demonstrate that plasma membranes derived from these cells are not very efficient in activation compared with the parental HT1080 cell line (Fig. 3, lane2). However, the addition of carefully titrated amounts of TIMP-2 to the extracts from p2AHT2a cells (Fig. 3, lanes 3-6) stimulated the conversion of proenzyme into the activated form, while further increase of TIMP-2 concentration (Fig. 3, lanes 7-9) caused complete inhibition of the reaction as was demonstrated earlier(22) . Conversely, the addition of increasing amounts of affinity-purified anti-TIMP-2 antibodies to the activation reaction prevents conversion of the 72T4Cl into activated forms (Fig. 4). The results of these experiments demonstrate that in addition to the carboxyl-terminal domain of the enzyme, the membrane-associated TIMP-2, represented by the 44-kDa cross-linking product with FLAG-CT is an essential element in the assembly of the membrane-associated activation complex. We therefore investigated the cross-linking pattern of I-TIMP-2 to the proteins of the plasma membranes from HT1080 and p2AHT2a cells (Fig. 5). Cross-linking of radiolabeled TIMP-2 to membrane extracts from either cell type yielded a specific product with an apparent molecular mass of 82 kDa (Fig. 5, lanes4-6 and 9-12), the formation of which was competitively inhibited by an excess of unlabeled TIMP-2 (Fig. 5, lanes 6-8 and 13), suggesting that a 60-kDa membrane-associated protein can interact with TIMP-2. Taken together with the results of cross-linking experiments using I-labeled 26-kDa CT (Fig. 2, 105- and 44-kDa specific products), these findings indicate a possibility that the carboxyl-end domain of 72T4Cl interacts with membrane-associated TIMP-2 (44-kDa cross-linking product), which in turn interacts with a putative 60-kDa membrane activator to make a tri-molecular activation complex represented by a 105-kDa specific cross-linking product.


Figure 3: Effect of TIMP-2 on activation of 72T4Cl by plasma membranes isolated from p2AHT2A cells. The 72T4Cl (15 ng) was incubated for 2 h alone (lane1) or activated with p2AHT2A plasma membranes (30 µg of protein, lanes2-9). Purified TIMP-2 (3, 10, 15, 30, 60, 120, and 240 ng) was added to the reaction (lanes3-9, respectively), and the mixture was incubated an additional 30 min at 0 °C prior to the addition of the enzyme.




Figure 4: Inhibition of 72T4Cl activation by affinity-purified anti-TIMP-2 antibodies. The 72T4Cl (15 ng) was incubated for 2 h at 37 °C in 25 mM HEPES-KOH buffer, pH 7.5, containing 0.1 mM CaCl(2), alone (lane1) or with HT1080 plasma membranes (8 µg, lanes2-8). Affinity-purified anti-TIMP-2 rabbit antibodies were added to the samples in lanes3-8 in the amounts indicated.




Figure 5: Cross-linking of radiolabeled TIMP-2 to plasma membrane extracts from phorbol 12-myristate 13-acetate-treated HT1080 and p2AHT2a cells. The I-labeled TIMP-2 (3 times 10^5 cpm, 4 times 10^6 cpm/µg, lanes1-13) in 25 mM HEPES-KOH buffer, pH 7.5, containing 150 mM KCl and 0.1 mM CaCl(2) was incubated with Lubrol (1%) extracts of plasma membranes from p2AHT2a cells (20 µg of protein, lane4; 40 µg of protein, lanes3, 5, 6, 7, and 8) or from TPA-induced p2AHT2a cells (20 µg, lane11; 40 µg, lane 12) or from TPA-induced HT1080 cells plasma membranes (20 µg, lanes9 and 13 or 40 µg, lane 10) with (lanes2 and 4-13) or without (lanes1 and 3) 0.5 mM of BS^3 for 1 h at 37 °C. Unlabeled ligand, TIMP-2, was added to the reaction for competition (10 ng, lane6; 30 ng, lane7; 100 ng, lanes8 and 13). After incubation, the reactions were analyzed by SDS-PAGE under reducing conditions, followed by autoradiography.



The fact that an excess of TIMP-2 inhibits the reaction suggests that assembly of such a complex may be blocked when both the activator and the carboxyl-end domain of the proenzyme are occupied with the inhibitor. Two alternative possibilities exist regarding the assembly of such complex. Either free putative activator binds TIMP-2 and then inhibitor-free enzyme sequentially or it can bind a preformed enzyme-inhibitor complex. We previously demonstrated (22) that at best only partial activation of a purified stoichiometric enzyme-inhibitor complex can be achieved in the presence of plasma membranes from HT1080 cells, and this residual activation can be abolished in the presence of additional amounts of TIMP-2. An examination of the activation reaction using TIMP-2-deficient p2AHT2a plasma membranes revealed that addition of a titrated amount of TIMP-2 prior to the addition of the enzyme improves the activity of these membranes (Fig. 4), while the results presented in Fig. 6show that a preformed enzyme-inhibitor complex is still resistant to activation. These results favor the sequential model of complex assembly in which membrane-bound free activator interacts with TIMP-2 prior to binding of the free enzyme for activation to occur.


Figure 6: Plasma membrane activation of 72T4Cl and the 72T4ClbulletTIMP-2 complex. The 72T4Cl (15 ng, lanes1-9) or 72T4ClbulletTIMP-2 complex (lanes 10-18) were incubated in 25 mM HEPES-KOH buffer, pH 7.5, containing 0.1 mM CaCl(2) alone (lanes1 and 10) or with plasma membranes from either HT1080 (20 µg, lanes2-5 and 11-14) or p2AHT2A cells (40 µg, lanes6-9 and 15-18) for the indicated times. An extra amount of TIMP-2 (50 ng) was added prior to the addition of plasma membranes to the samples in lanes5, 9, 14, and 18. The 72T4ClbulletTIMP-2 stoichiometric complex was prepared from purified TIMP-2 and 72T4Cl and separated by gel filtration chromatography as previously described(22) .



The Cell Surface Activator Serves As a Receptor for the Inhibitor TIMP-2, and the Resulting Complex in Turn Acts as a Receptor for 72T4Cl

To verify the interpretation of the cross-linking and activation experiments presented above, we have determined the kinetic parameters and interdependence of TIMP-2 and 26-kDa CT binding to the surface of HT1080 and p2AHT2a cells ( Fig. 7and 8). The use of the carboxyl-end domain of 72T4Cl is advantageous in these experiments compared with the use of pro-enzyme, since the latter contains additional binding domains (32, 36, 37) that are capable of binding extracellular matrix macromolecules produced by the cells in culture as well as present in the media. The results presented in Fig. 7show a saturable and specific binding of TIMP-2 and 72T4Cl carboxyl-end domain to the cell surface with K(d) 2.54 times 10 and 0.56 times 10, respectively. The binding of both ligands to the cell surface of HT1080 cells is induced by treatment with TPA up to 40,000 and 7000 binding sites for TIMP-2 and FLAG-CT per TPA-induced HT1080 cell, respectively. Binding of these ligands to the cell surface of p2AHT2a cells have identical affinities, with the number of sites per cell considerably reduced and comparable with those of non-induced HT1080 cells (not shown). Treatment of the p2AHT2a cells with TPA does not increase binding of either ligand. This is in agreement with our previous observation that the TPA induction pathway in these cells is blocked(29) .


Figure 7: Binding of radiolabeled TIMP-2 and FLAG-26-kDa CT fusion to HT1080 and p2AHT2a cells. The cells were washed with serum-free media containing 25 mM HEPES buffer, pH 7.3, and 1 mg/ml bovine serum albumin and incubated on ice for 20 min with (background) or without unlabeled TIMP-2 or FLAG-CT fusion ligands prior to addition of I-radiolabeled TIMP-2 or FLAG-CT to reach indicated concentrations. The cells were incubated for 40 min on ice, washed five times with serum-free media, and lysed in 0.4 M NaOH, 0.3% SDS; radioactivity was then counted. One well of each cluster was used to count the number of cells. The nonspecific background binding was determined using a 500-fold excess of unlabeled over highest concentration of I ligand. The binding is displayed as a function of ligand concentration. The solidcurves are the binding curves determined by a fit to the Scatchard equation. B and C, the Scatchard plots of data presented in A. The dissociation constants and saturation binding were obtained from a least squares (solidline) fit to the data.



If, as proposed above, the binding of the carboxyl-end domain of the enzyme represented by the 105-kDa specific cross-linking product is mediated through membrane-associated TIMP-2, then such interdependent interaction of two ligands with the cell surface can be described by the following model.

A membrane-associated activator (A) acts as a receptor for exogenous or endogenous TIMP-2 (T). The activator-TIMP-2 complex (AT) in turn acts as a receptor for 72T4Cl (C) to form the complex ATC, which causes activation of the enzyme. The latter binding is mediated through the interaction between the carboxyl-end domain of 72T4Cl and TIMP2 present in the AT complex and has the same dissociation constant as that of the pro-72T4Cl-TIMP-2 complex (38) in solution. These steps are described by the following reactions where lower and uppercasesymbols represent free and total concentrations of each reactant, respectively,

where at equilibrium, K(1) = abullett/at, K(2) = atbulletc/atc, and K(3) = tbulletc/tc. Solutions of the , , and for at and atc (total cell surface-bound 26-kDa CT) in terms of c and t are

assuming that K(2) = K(3), total cell surface-bound TIMP2 is

where c and t are calculated by

The dissociation constants of TIMP-2 and the 26-kDa CT for the HT1080 cell surface, K(1) and K(2), were determined above (Fig. 7). The constant K(3) is the dissociation constant of the CT complex in solution that is identical to K(2) ( Fig. 7and (38) ) within error of measurement. The total number of binding sites for both ligands were determined independently in this experiment (not shown) since the total number of such sites and the ratio of occupied (AT) to unoccupied (A) receptor can vary significantly between experiments. This is due to the fact that TPA induction stimulates the expression of receptor A (Fig. 7) while inhibiting the expression of TIMP-2(39) .^2

and were used to model the competition kinetics of I-labeled 26-kDa CT cell surface binding by exogenously added TIMP-2 (Fig. 8A, solidline) and I-TIMP-2 binding by exogenously added 26-kDa CT (Fig. 8B, solidline), respectively. The experimental data (open and solidsquares) obtained in such competition experiments are in good agreement with computer-generated predictions (solidlines) and thus support a tri-molecular complex model outlined above.


Figure 8: Competition for binding of the radiolabeled FLAG-CT fusion with TIMP-2 (A) and the radiolabeled TIMP-2 with FLAG-CT fusion (B). The competition of binding of radiolabeled ligands with the unlabeled counterpart were performed as described in Fig. 7. The cells were preincubated with increasing concentrations of unlabeled ligand prior to addition of I-labeled 72-kDa CT (2.2 times 10M) or I-TIMP-2 (4.4 times 10M). Competition of I-labeled 26-kDa CT with TIMP-2 (A) and of I-TIMP-2 with 26-kDa CT (B) were performed in the same experiment. Binding is displayed as a function of competing ligand concentration. The solidlines are the computer-generated model predictions, as described in the text.



The pattern of I-labeled 26-kDa CT binding and competition with TIMP-2 suggest the presence of membrane-bound activator in two forms, a free form available for binding of TIMP-2 and 72T4Cl and a form occupied with endogenous TIMP-2 that can bind 72T4Cl only. Availability of the first form on the surface of TPA-induced HT1080 cells explains the increase in binding of 26-kDa CT in the presence of small quantities of exogenously labeled TIMP-2, which is necessary for the 26-kDa CT to bind to free activator. Further increase in the concentration of TIMP-2 leads to complete inhibition of binding due to the formation of the 26-kDa CTbulletTIMP-2 and activatorbulletTIMP-2 complexes. The competition experiments described here do not discriminate as to whether the assembly of the ATC complex can be accomplished by binding of TC to A. To determine whether this reaction takes place, we conducted a separate binding experiment using a preformed CT complex with a radiolabeled TIMP-2 component and demonstrated that the K(d) of its binding to cell surface is 3.7 times lower then that of TIMP-2 alone in the same experiment (data not shown). This result can explain why the 72T4ClbulletTIMP-2 complex is resistant to activation in most cases and is activated very slowly in others (see ``Discussion'' and (26) ).

Purification of a Cell Surface Activator in Complex with TIMP-2 and Carboxyl-end Domain of 72T4Cl

The results of the activation, cross-linking ( Fig. 2and Fig. 5), and cell binding experiments ( Fig. 7and Fig. 8) provided a rationale for purification of the putative activator and positive identification of the 21-kDa membrane-associated component, presumably TIMP-2, that produces a 44-kDa specific cross-link with 26-kDa CT. Since cell surface binding of 26-kDa CT is dependent on the presence of the activatorbulletTIMP-2 complex, we attempted an isolation of the putative activator using an affinity column with immobilized recombinant FLAG-CT. To avoid chemical modifications that may inactivate the fragment (see above and (22) ), we used an anti-FLAG-M1 Ca-dependent monoclonal antibody affinity column to immobilize the FLAG-CT fusion protein in an oriented fashion. A complex of anti-FLAG-M1bulletFLAG-CTbulletTIMP-2-activator is expected to form and be eluted as a FLAG-CTbulletTIMP-2-activator complex upon chelating of Ca ions in the column buffer with EDTA.

The anti-FLAG-M1 antibody resin (0.5-ml bed volume) was saturated with an excess of purified recombinant FLAG-CT fusion protein. The Lubrol extract of HT1080 membranes (100 mg, total protein) was applied on the FLAG-CT-M1 column, and bound proteins were eluted in 25 mM HEPES buffer, pH 7.5, containing 2 mM EDTA. The SDS-PAGE analysis of the eluted material (Fig. 9) revealed the presence of four protein bands. In addition to protein bands corresponding to the FLAG-CT and the 21-kDa band co-migrating with the TIMP-2, two bands, major and minor, with a molecular mass of about 60 kDa, are apparent on the gel. These proteins were electroblotted onto a polyvinylidene difluoride membrane and subjected to amino-terminal sequence analysis. The 21-kDa protein had the amino-terminal sequence XSXSPVHPQQAFXNADVVI, identifying this protein as TIMP-2 (the corresponding TIMP-2 sequence is C(1)SCSPVHPQQAFCNADVVIRA). The amino-terminal amino acid sequences of the major and minor 60-kDa bands were identical. This sequence (Fig. 9B) identified a novel protein with substantial homology to sequences of secreted metalloproteases. No identical sequence was found in a GeneBank search. While this work was in progress, Sato et al.(28) isolated a cDNA clone for MT-MMP from human placenta RNA using degenerate polymerase chain reaction priming. The isolated cDNA upon transfection into Cos-1 cells caused activation of the co-transfected 72T4Cl(28) . The sequence of the 60-kDa putative metalloprotease isolated in a tri-molecular complex with 26-kDa CT and TIMP-2 in our experiments is identical to the sequence of MT-MMP (residues 111-130) and thus represents its activated form with a loss of 105 amino acid residues of the propeptide, including the conserved propeptide Cys residue.


Figure 9: Purification of the activator in complex with TIMP-2 and FLAG-CT fusion from Lubrol extract of TPA-induced HT1080 cell plasma membranes. A, affinity chromatography of the membrane extract on FLAG-CT, immobilized on an M1 anti-FLAG monoclonal antibody column. A fresh portion of 0.5 ml of the M1 monoclonal antibody resin (IBI, 0.5-ml bed volume) in 10 mM HEPES buffer, pH 7.5, containing 150 mM KCl, 1 mM Ca, and 0.002% Brij 35, was saturated with 1 mg of purified FLAG-CT fusion protein and washed with 20 ml of the same buffer, containing 1% Lubrol. Plasma membranes from HT1080 cells in the above buffer were extracted with 1% Lubrol for 1 h at 0 °C. The extract (100 mg of protein) was loaded on the column, washed with 50 ml of the same buffer and 10 ml of the same buffer containing 0.1% Lubrol, and eluted with the same buffer containing 2 mM EDTA and no Ca. Ten fractions of 500 µl each were collected and analyzed by SDS-PAGE. Fractions 3 and 4 are shown (lanes1 and 2). B, alignment of amino-terminal sequence of the 60-kDa protease with known secreted metalloproteases.




DISCUSSION

Cell translocation during both normal and pathological processes of tissue remodeling requires spatially regulated degradation of the existing ECM macromolecules. Two proteolytic systems are apparently involved in this process: (i) plasminogen activator of the urokinase type, which is sequestered on the cell surface and is activated via interaction with its receptor(40, 41, 42, 43, 44, 45, 46, 47, 48, 49) and (ii) secreted metalloproteases that initiate degradation of collagens and proteoglycans(1, 2, 3, 4, 5, 6) . All known members of the latter group of enzymes are secreted as soluble zymogens and require activation in the extracellular space. Their activation can be accomplished by a proteolytic cascade initiated by serine proteases such as plasmin that invariably leads to processing of the amino-terminal pro-peptide of the enzyme(7, 8, 9, 10, 11, 12, 13, 14) . This causes a structural transition unlocking the active center of the partially processed enzyme by the cysteine switch mechanism (11) and is followed by further processing of the amino terminus by auto-proteolysis or with the help of a related metalloprotease such as stromelysin(8, 10, 12, 13) . Although activation mechanisms in solution have been worked out in considerable detail, the mechanistic understanding of spatial regulation of metalloprotease activity has remained an elusive goal.

Recently, we (22) and others (23, 24, 25, 26, 27) demonstrated a cell membrane-dependent mechanism of activation specific for 72T4Cl. In the presence of plasma membranes from TPA-induced HT1080 cells, the activation of exogenously added, inhibitor-free 72T4Cl generates an active enzyme of 62 kDa. Formation of the 72T4ClbulletTIMP-2 complex inhibits activation at the level of the initial pro-peptide cleavage(22) . Membrane-dependent activation of 72T4Cl is competitively inhibited in the presence of a 26-kDa peptide derived from the carboxyl domain of the enzyme (22) and the inhibitor, TIMP-2, which forms a complex with the same domain. These results were interpreted to mean that interaction of the carboxyl-end domain with a cell membrane-associated protein is essential for activation of the enzyme.

Cross-linking and activation experiments described in this report demonstrate that membrane-associated TIMP-2 interacts with the carboxyl-end domain of 72T4Cl and is an essential part of the cell surface activation complex. Cross-linking of radiolabeled FLAG-CT with extracts of cell membrane yielded two specific products with molecular mass of 105 and 44 kDa, the latter representing the cross-link with TIMP-2. Chemical modifications abolishing the interaction of the carboxyl-end domain with the inhibitor also abolish its ability to competitively inhibit membrane activation and the appearance of the 44-kDa cross-linking product. The activity of plasma membranes from p2AHT2a cells that do not secrete appreciable amounts of TIMP-2 can be improved by an addition of titrated amounts of the purified inhibitor. Finally, anti-TlMP-2 antibodies inhibited the activation of the 72T4Cl. Cross-linking of radiolabeled TIMP-2 with either preparation of cell membranes yielded an 82-kDa specific product. These results suggested that activation of 72T4Cl requires an assembly of a tri-molecular stoichiometric complex represented by the 105-kDa specific cross-link and involving a 60-kDa membrane-associated activator, the inhibitor, TIMP-2, and the carboxyl-end domain of 72T4Cl.

This model implies that the enzyme does not bind to the cell surface independently of TIMP-2. The binding experiments presented here support this model. Kinetic equations describing this model predict competition curves that are in agreement with the experimental data. The results demonstrate the presence of occupied and unoccupied receptor of TIMP-2 on the cell surface of the TPA-induced HT1080 cells. The occupied form can serve as a receptor for the carboxyl-end domain of 72T4Cl, the binding of which increases in the presence of small amounts of TIMP-2, as the unoccupied receptor becomes saturated with the inhibitor. Further increase of the TIMP-2 concentration completely inhibits the binding of the 26-kDa CT since the receptor saturated with TIMP-2 fails to bind the 26-kDa CTbulletTIMP-2 complex. Thus, the competition experiments demonstrate the existence of TIMP-2-dependent carboxyl-end domain binding site but do not exclude the possibility of a TIMP-2-independent 26-kDa CT receptor. The linearity of Scatchard plots for FLAG-CT binding, however, suggests no distinguishable second binding component.

The model presented here can explain the apparent discrepancy between the results of several laboratories showing that TIMP-2 inhibits activation of 72T4Cl (22, 23) and the observation of Brown et al.(26) where the radiolabeled complex of 72T4ClbulletTIMP-2 was activated using concanavalin A-treated fibroblasts. In the former case, relatively short incubation times (1 h) were employed, while long incubation times (22 h) were used to observe the latter. Thus, while inhibition of activation of 72T4Cl in complex with TIMP-2 was not observed during short incubations, longer incubations could allow for rearrangement of components to occur, producing the observed activation. Our results are also in agreement with the cell surface binding experiments using 72T4Cl observed by Emonard et al.(50) in two human breast adenocarcinoma cells, MDA-MB231 and MCF-7, although some what lower affinity (K(d) = 2 times 10M) was observed in these experiments.

Purification of the activator was achieved here by isolating its occupied form in a tri-molecular complex with the recombinant FLAG-CT fusion protein. The purified complex contained the carboxyl-end domain fragment, TIMP-2, and a 60-kDa protein whose amino-terminal sequence is homologous to sequences of other secreted metalloproteases. The isolated preparation of such a complex can not be used in cross-linking experiments to demonstrate specificity of interaction since all the involved components are present in a ratio defined by the properties of the affinity column. The activation assay is also not feasible for the same reason. To positively demonstrate activity of the isolated protease, transfection experiments with its cDNA clone are necessary. While this work was in progress, Sato et al.(28) isolated a cDNA clone of a membrane-associated metalloprotease, MT-MMP, using degenerate polymerase chain reaction primers, that is identical to the activator isolated here. Co-transfection of the expression vectors with this cDNA clone together with cDNA of 72T4Cl caused activation of the expressed enzyme, proving that MT-MMP is indeed the 72T4Cl activator.

The protein sequence of the 60-kDa protease isolated here is identical to the sequence of the MT-MMP, positions 111-128, demonstrating that this protein is an activated form of MT-MMP activator, thus posing the question: how is the activator activated? The likely answer to this question lies in the amino acid sequence Arg-Arg-Lys-Arg just upstream from the amino terminus (Ala) of the activated MT-MMP that makes the pro-peptide an excellent substrate for serine proteases such as plasmin or plasminogen activator. This sequence motif is also found in other members of the metalloprotease family, such as collagenase and stromelysin, that can be activated by plasmin. This sequence is absent from the pro-peptide of 72T4Cl, which is resistant to plasmin activation. Therefore, it is very likely that activation of the 72T4Cl is the result of a cell surface proteolytic cascade that involves activation of the membrane metalloprotease by the membrane-associated plasmin (51, 52) and/or plasminogen activator, which itself is activated upon binding to a specific receptor. Activated MT-MMP serves as a receptor for TIMP-2 forming a complex that binds 72T4Cl, leading to its activation.

The data presented here cannot distinguish with certainty whether binding of TIMP-2 requires prior activation of MT-MMP or if the inhibitor can bind the proenzyme, as in the case of 72T4Cl. It is also not clear whether the inhibitor binds to an activated form of MT-MMP through the amino-terminal inhibitory domain (53, 54, 55) while exposing the carboxyl-end domain for interaction with 72T4Cl. We have recently described (13) the isolation of a similar complex between 92T4CI, TIMP, and activated interstitial collagenase(13) , where the activity of the latter was inhibited. Whether MT-MMP retains the proteolytic activity while in complex with TIMP-2 and the carboxyl-end domain of 72T4Cl remains to be elucidated.

The implications of the cell surface activation cascade of 72T4Cl for mechanisms of tissue remodeling are far reaching. 72T4Cl as well as TIMP-2 could be synthesized by a different set of cells than those that express the MT-MMP and utilize the membrane-bound 72T4Cl to accomplish cell translocation. Such a mechanism creates another level of regulation of cell migration in a spatially organized fashion. This model is supported by recent observations (56, 57, 58, 59) that, in most cases, soluble secreted metalloproteases are synthesized in the tumor stroma, but, as in the case of the breast cancer, the enzyme is associated with an epithelial component of the tumor. Moreover, the involvement of TIMP-2 in activation of 72T4Cl can explain a surprising observation of Visscher et al.(60) , where a poor prognosis in breast cancer is correlated with the presence of TIMP-2 in the tumors rests rather than with the amount of 72T4Cl protein. Thus, the model of the 72T4Cl activation described here presents a realistic target for intervention with metastatic disease and other pathological processes of tissue remodeling.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1 AR39472 and RO1 AR40618 and Training Grant T32 AR07284 and Monsanto Co./Washington University Biomedical Research Agreement. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: General Atomics Inc., Biosciences Bldg. 2, 3550 General Atomics Ct., P. O. Box 85608, San Diego, CA 92186-9784.

(^1)
The abbreviations used are: ECM, extracellular matrix; TIMP-1 and TIMP-2, tissue inhibitors of metalloproteases 1 and 2, respectively; 92T4Cl and 72T4Cl, 92- and 72-kDa type IV collagenase, respectively; 26-kDa CT, 26-kDa carboxyl-terminal fragment of 72T4Cl; FLAG-CT, recombinant fusion protein of 26-kDa CT with FLAG peptide; MT-MMP, membrane-type matrix metalloprotease; PAGE, polyacrylamide gel electrophoresis; TPA, 12-O-tetradecanoyl-phorbol acetate; BS^3, bis(sulfosuccinimidyl) suberate.

(^2)
A. Y. Strongin, I. Collier, G. Bannikov, B. L. Marmer, G. A. Grant, and G. I. Goldberg, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Arthur Eisen, Rafael Kophan, and Sergey Troyanovsky for critical review of the manuscript.


REFERENCES

  1. Goldberg, G. I., and Eisen, A. Z. (1991) in Regulatory Mechanisms in Breast Cancer (Lippman, M., and Dixon, R., eds) pp. 421-440, Kluwer Academic Press, Boston
  2. Birkedal-Hansen, H., Moore, W. G., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., and Engler, J. A. (1993) Crit. Rev. Oral Biol. Med. 4, 197-250 [Abstract]
  3. Matrisian, L. M. (1992) Bioessays 14, 455-463 [Medline] [Order article via Infotrieve]
  4. Liotta, L. A., Steeg, P. S., and Stetler-Stevenson, W. G. (1991) Cell 64, 327-336 [Medline] [Order article via Infotrieve]
  5. Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L. A. (1993) Annu. Rev. Cell Biol. 9, 541-573 [CrossRef]
  6. Stetler-Stevenson, W. G., Liotta, L. A., and Kleiner, D. E., Jr. (1993) FASEB J. 7, 1434-1441 [Abstract/Free Full Text]
  7. Grant, G. A., Eisen, A. Z., Marmer, B. L., Roswit, W. T., and Goldberg, G. I. (1987) J. Biol. Chem. 262, 5886-5889 [Abstract/Free Full Text]
  8. Ogata, Y., Enghild, J. J., and Nagase, H. (1992) J. Biol. Chem. 267, 3581-3584 [Abstract/Free Full Text]
  9. Nagase, H., Suzuki, K., Morodomi, T., Enghild, J. J., and Salvesen, G. (1992) Matrix (Suppl.) 1, 237-244 [Medline] [Order article via Infotrieve]
  10. Knauper, 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]
  11. Van Wart, H. E., and Birkedal-Hansen, H. (1990) Proc. Natl. Acad. Sci U. S. A. 87, 5578-5582 [Abstract]
  12. He, C., Wilhelm, S. M., Pentland, A. P., and Goldberg, G. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2632-2636 [Abstract]
  13. Goldberg, G. I., Strongin, A., Collier, I. E., Genrich, L. T., and Marmer, B. L. (1992) J. Biol. Chem. 267, 4583-4591 [Abstract/Free Full Text]
  14. Kleiner, D. E., Jr., and Stetler-Stevenson, W. G. (1993) Curr. Opin. Cell Biol. 5, 891-897 [Medline] [Order article via Infotrieve]
  15. Welgus, H. G., Stricklin, G. P., Eisen, A. Z., Bauer, E. A., Cooney, R. V., and Jeffrey, J. J. (1979) J. Biol. Chem. 254, 1938-1943 [Medline] [Order article via Infotrieve]
  16. Stricklin, G. P., and Welgus, H. G. (1983) J. Biol. Chem. 258, 12252-12258 [Abstract/Free Full Text]
  17. Docherty, A. J., Lyons, A., Smith, B. J., Wright, E. M., Stephens, P. E., Harris, T. J., Murphy, G., and Reynolds, J. J. (1985) Nature 318, 66-69 [Medline] [Order article via Infotrieve]
  18. Carmichael, D. F., Sommer, A., Thompson, R. C., Anderson, D. C., Smith, C. G., Welgus, H. G., and Stricklin, G. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2407-2411 [Abstract]
  19. Stetler-Stevenson, W. G., Krutzsch, H. C., and Liotta, L. A. (1989) J. Biol. Chem. 264, 17374-17378 [Abstract/Free Full Text]
  20. Goldberg, G. I., Marmer, B. L., Grant, G. A., Eisen, A. Z., Wilhelm, S. M., and He, C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8207-8211 [Abstract]
  21. Boone, T. C., Johnson, M. J., De Clerck, Y. A., and Langley, K. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2800-2804 [Abstract]
  22. Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol. Chem. 268, 14033-14039 [Abstract/Free Full Text]
  23. Ward, R. V., Atkinson, S. J., Slocombe, P. M., Docherty, A. J., Reynolds, J. J., and Murphy, G. (1991) Biochim. Biophys. Acta 1079, 242-246 [Medline] [Order article via Infotrieve]
  24. Azzam, H. S., and Thompson, E. W. (1992) Cancer Res. 52, 4540-4544 [Abstract]
  25. Murphy, G., Willenbrock, F., Ward, R. V., Cockett, M. I., Eaton, D., and Docherty, A. J. (1992) Biochem. J. 283, 637-641 [Medline] [Order article via Infotrieve]
  26. Brown, P. D., Kleiner, D. E., Unsworth, E. J., and Stetler-Stevenson, W. G. (1993) Kidney Int. 43, 163-170 [Medline] [Order article via Infotrieve]
  27. Seltzer, J. L., Lee, A. Y., Akers, K. T., Sudbeck, B., Southon, E. A., Wayner, E. A., and Eisen, A. Z. (1994) Exp. Cell Res. 213, 365-374 [CrossRef][Medline] [Order article via Infotrieve]
  28. 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]
  29. Frisch, S. M., Reich, R., Collier, I. E., Genrich, T. L., Martin, G., and Goldberg, G. I. (1990) Oncogene 5, 75-83 [Medline] [Order article via Infotrieve]
  30. Goldberg, G. I., Wilhelm, S. M., Kronberger, A. M., Bauer, E. A., Grant, G. A., and Eisen, A. Z. (1986) J. Biol. Chem. 261, 6600-6605 [Abstract/Free Full Text]
  31. Gorman, C. (1985) in DNA Cloning (Glover, D. M., ed) Vol. 2, pp. 143-170, IRL Press, Oxford
  32. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He, C., Bauer, E. A., and Goldberg, G. I. (1988) J. Biol. Chem. 263, 6579-6587 [Abstract/Free Full Text]
  33. Oka, T., Sakamoto, S., Miyoshi, K., Fuwa, T., Yoda, K., Yamasaki, M., Tamura, G., and Miyake, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7212-7216 [Abstract]
  34. Karelina, T. V., Hruza, G. J., Goldberg, G. I., and Eisen, A. Z. (1993) J. Invest. Dermatol. 100, 159-165 [Abstract]
  35. Fridman, R., Fuerst, T. R., Bird, R. E., Hoyhtya, M., Oelkuct, M., Kraus, S., Komarek, D., Liotta, L. A., Berman, M. L., and Stetler-Stevenson, W. G. (1992) J. Biol. Chem. 267, 15398-15405 [Abstract/Free Full Text]
  36. Collier, I. E., Krasnov, P. A., Strongin, A. Y., Birkedal-Hansen, H., and Goldberg, G. I. (1992) J. Biol. Chem. 267, 6776-6781 [Abstract/Free Full Text]
  37. Strongin, A. Y., Collier, I. E., Krasnov, P. A., Genrich, L. T., Marmer, B. L., and Goldberg, G. I. (1993) Kidney Int. 43, 158-162 [Medline] [Order article via Infotrieve]
  38. Howard, E. W., and Banda, M. J. (1991) J. Biol. Chem. 266, 17972-17977 [Abstract/Free Full Text]
  39. Stetler-Stevenson, W. G., Brown, P. D., Onisto, M., Levy, A. T., and Liotta, L. A. (1990) J. Biol. Chem. 265, 13933-13938 [Abstract/Free Full Text]
  40. Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund, L. R., Dano, K., Appella, E., and Blasi, F. (1990) EMBO J. 9, 467-474 [Abstract]
  41. Bianchi, E., Cohen, R. L., Thor, A. T., Todd, R. F., III, Mizukami, I. F., Lawrence, D. A., Ljung, B. M., Shuman, M. A., and Smith, H. S. (1994) Cancer Res. 54, 861-866 [Abstract]
  42. Crowley, C. W., Cohen, R. L., Lucas, B. K., Liu, G., Shuman, M. A., and Levinson, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5021-5025 [Abstract]
  43. Pyke, C., Graem, N., Ralfkiaer, E., Ronne, E., Hoyer-Hansen, G., Brunner, N., and Dano, K. (1993) Cancer Res. 53, 1911-1915 [Abstract]
  44. Pyke, C., Kristensen, P., Ralfkiaer, E., Grondahl-Hansen, J., Eriksen, J., Blasi, F., and Dano, K. (1991) Am. J. Pathol. 138, 1059-1067 [Abstract]
  45. Ossowski, L. (1992) Cancer Res. 52, 6754-6760 [Abstract]
  46. Ossowski, L., Clunie, G., Masucci, M. T., and Blasi, F. (1991) J. Cell Biol. 115, 1107-1112 [Abstract]
  47. Quax, P. H., Pedersen, N., Masucci, M. T., Weening-Verhoeff, E. J., Dano, K., Verheijen, J. H., and Blasi, F. (1991) Cell Regul. 2, 793-803 [Medline] [Order article via Infotrieve]
  48. Fazioli, F., and Blasi F. (1994) Trends Pharmacol. Sci. 15, 25-29 [CrossRef][Medline] [Order article via Infotrieve]
  49. Montgomery, A. M., De Clerck, Y. A., Langley, K. E., Reisfeld, R. A., and Mueller, B. M. (1993) Cancer Res. 53, 693-700 [Abstract]
  50. Emonard, H. P., Remacle, A. G., Noel, A. C., Grimaud, J. A., Stetler-Stevenson, W. G., and Foidart, J. M. (1992) Cancer Res. 52, 5845-5848 [Abstract]
  51. Meissauer, A., Kramer, M. D., Schirrmacher, V., and Brunner, G. (1992) Exp. Cell Res. 199, 179-190 [Medline] [Order article via Infotrieve]
  52. Kwaan, H. C. (1992) Cancer Metastasis Rev. 11, 291-311 [Medline] [Order article via Infotrieve]
  53. Miyazaki, K., Funahashi, K., Numata, Y., Koshikawa, N., Akaogi, K., Kikkawa, Y., Yasumitsu, H., and Umeda, M. (1993) J. Biol. Chem. 268, 14387-14393 [Abstract/Free Full Text]
  54. Murphy, G., Houbrechts, A., Cockett, M. I., Williamson, R. A., O'Shea, M., and Docherty, A. J. (1991) Biochemistry 30, 8097-8102 [Medline] [Order article via Infotrieve]
  55. DeClerck, Y. A., Yean, T. D., Lee, Y., Tomich, J. M., and Langley, K. E. (1993) Biochem. J. 289, 65-69 [Medline] [Order article via Infotrieve]
  56. Polette, M., Clavel, C., Cockett, M., Girod de Bentzmann, S., Murphy, G., and Birembaut, P. (1993) Invasion Metastasis 13, 31-37 [Medline] [Order article via Infotrieve]
  57. Tryggvason, K., Hoyhtya, M., and Pyke, C. (1993) Breast Cancer Res Treat. 24, 209-218 [Medline] [Order article via Infotrieve]
  58. Poulsom, R., Pignatelli, M., Stetler-Stevenson, W. G., Liotta, L. A., Wright, P. A., Jeffery, R. E., Longcroft, J. M., Rogers, L., and Stamp, G. W. (1992) Am. J. Pathol. 141, 389-396 [Abstract]
  59. Pyke, C., Ralfkiaer, E., Tryggvason, K., and Dano, K. (1993) Am. J. Pathol. 142, 359-365 [Abstract]
  60. Visscher, D. W., Hoyhtya, M., Ottosen, S. K., Liang, C.-M., Sarcar, F. H., Crissman, J. D., and Fridman, R. (1994) Int. J. Cancer 58, 1-6 [Medline] [Order article via Infotrieve]

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