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Intact Vitronectin Induces Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinases-2 Expression and Enhanced Cellular Invasion by Melanoma Cells

Intact Vitronectin Induces Matrix Metalloproteinase-2 and Tissue Inhibitor of... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 1, Issue of January 2, pp. 143–149, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Intact Vitronectin Induces Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinases-2 Expression and Enhanced Cellular Invasion by Melanoma Cells* (Received for publication, October 11, 1996, and in revised form, September 25, 1997) Lisa M. Bafetti‡, Timothy N. Young§, Yoshifumi Itoh‡, and M. Sharon Stack‡¶i From the Departments of ‡Obstetrics & Gynecology and ¶Cell & Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611 and §Washington University School of Medicine, St. Louis, Missouri 63110 The initial site of melanoma cell metastasis is fre- adhesion to the regional lymph nodes, which correlates with poor prognosis, is mediated via interaction of specific integrins quently the regional lymph nodes, and the appearance of lymph node metastasis correlates with poor progno- on the melanoma cell surface with lymph node vitronectin (3). sis. Lymph node adhesion is mediated by an interaction Previous data have demonstrated a relationship between ele- avb3 and lymph node between the tumor cell integrin vated levels of vitronectin-binding integrins and increased mel- vitronectin. In this study, we explored the relationship anoma cell invasiveness (4 – 6). Furthermore, ligation of the between adhesion and proteolysis by examining the di- avb3 integrin on melanoma cells by anti-avb3 antibodies en- rect effect of vitronectin receptor ligation on matrix hances secretion of matrix metalloproteinase-2 (MMP-2, gela- metalloproteinase-2 (MMP-2) production by B16F1 and tinase A, 72-kDa type IV collagenase), resulting in increased B16F10 melanoma cells. We report a dose-dependent in- cellular invasiveness (7). Together, these data suggest that inte- crease in secretion of both MMP-2 and tissue inhibitor of grin-mediated binding of tumor cells to a specific matrix-associ- metalloproteinases-2 (TIMP-2) in response to vitronec- ated protein, such as vitronectin, can promote tumor cell inva- tin. Cellular invasiveness was also enhanced by sion by increasing the levels of a matrix-degrading proteinase. vitronectin, as shown by the increased ability of The majority of integrins recognize multiple extracellular vitronectin-treated cells to invade a synthetic basement matrix ligands (1, 2), and precise biologic responses may be membrane (Matrigel). Both the vitronectin-induced regulated by differential integrin ligation with distinct extra- MMP-2 production and vitronectin-enhanced invasion cellular matrix proteins. In this study, we have explored the were blocked by the peptide ligand Arg-Gly-Asp-Ser direct effect of the matrix-associated ligand vitronectin on pro- (RGDS). Furthermore, neither plasmin-degraded duction of MMP-2 by melanoma cells. We report a dose-depend- vitronectin nor the peptide ligand RGDS stimulated ent increase in secretion of both MMP-2 and tissue inhibitor of MMP-2 secretion or invasiveness, indicating that a mul- metalloproteinases-2 (TIMP-2), as well as enhanced cellular tivalent ligand-receptor interaction rather than simple invasiveness, in response to vitronectin. Intact vitronectin is receptor occupancy was required for MMP-2 induction. MMP-2 and MMP-2/TIMP-2 interaction with the plasma required for MMP-2 induction because neither plasmin-treated membrane of melanoma cells resulted in enhanced cat- vitronectin nor a peptide ligand (Arg-Gly-Asp-Ser (RGDS)) al- C-labeled gelatin, suggesting alytic activity against ters MMP-2 secretion, indicating the requirement for a multi- that membrane association may function in posttrans- valent ligand-receptor interaction. Furthermore, MMP-2 inter- lational regulation of MMP-2 activity. This is supported acts with the plasma membranes of melanoma cells, exhibits by data showing increased cellular invasion by cells enhanced catalytic activity relative to the solution phase en- containing membrane-bound MMP-2. Binding of zyme, and increases cellular invasive activity. Membrane bind- proMMP-2 and proMMP-2/TIMP-2 to melanoma cells ing of MMP-2 is unaffected by RGDS, and melanoma cell ad- was not inhibited by RGDS, and melanoma cell adhesion hesion to vitronectin is not inhibited by MMP-2, indicating that to vitronectin was unaffected by pro- or active MMP-2, MMP-2 does not bind the vitronectin receptor on murine mel- indicating that MMP-2 did not interact with the murine anoma cells. These data suggest a potential physiologic mech- vitronectin receptor. Together, these data provide evi- anism whereby the relative integrity of the adhesive substra- dence for a functional link between adhesion and pro- tum may differentially regulate secretion and activity of a teolysis and suggest a potential mechanism whereby matrix-degrading proteinase and subsequent cellular invasive adhesion of an invasive cell to the extracellular matrix behavior. regulates subsequent invasive behavior. EXPERIMENTAL PROCEDURES Cell Culture—The murine B16F10 and B16F1 melanoma cell lines Adhesion of tumor cells to specific extracellular matrix mac- were obtained from Dr. I. J. Fidler (The University of Texas M. D. romolecules is an initial component of the metastatic process Anderson Hospital) and were cultured in Eagle’s minimal essential (reviewed in Refs. 1 and 2). In metastatic melanoma, tumor cell medium supplemented with 5% fetal calf serum, nonessential amino acids, L-glutamine, and vitamins. Prior to each experiment, cells were washed with calcium and magnesium-free Dulbecco’s phosphate-buff- * This work was supported by Research Grant CA 58900 from the ered saline (PBS) and incubated for 2 min at 25 °C with 1 mM EDTA in NCI, National Institutes of Health (to M. S. S.). The costs of publication serum-free Eagle’s minimal essential medium to release cells from the of this article were defrayed in part by the payment of page charges. culture flask. Cells were seeded in 1 ml of serum-free Eagle’s minimal This article must therefore be hereby marked “advertisement”inac- essential medium at a density of 1 3 10 cells/ml and incubated 18 h at cordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: Northwestern Uni- versity Medical School, 303 East Chicago Ave., Tarry 4-751, Chicago, The abbreviations used are: MMP-2, matrix metalloproteinases-2; IL 60611. Tel.: 312-908-8216; Fax: 312-908-8773; E-mail: mss130@ TIMP, tissue inhibitor of metalloproteinases; PBS, phosphate-buffered anima.nums.nwu.edu. saline; APMA, amino-phenylmercuric acetate. This paper is available on line at http://www.jbc.org 143 This is an Open Access article under the CC BY license. 144 Vitronectin Induction of Matrix Metalloproteinase-2 37 °C in the presence of vitronectin, peptide, or both as described below. Adhesion Assay—Vitronectin was passively adsorbed to 24-well cul- ture plates as described previously (15), blocked by incubating with In some experiments, colchicine (0.2 mM) was included for inhibition of PBS containing 2% bovine serum albumin for2hat25°C,and utilized secretion. Conditioned media were removed and concentrated 4-fold at for cell attachment assays as described by Pierschbacher and Ruoslahti 4 °C using a Centricon 10 (Amicon) concentrator and analyzed imme- (16). Briefly, cells were plated at a constant of 1.5 3 10 cells/ml in a diately as described below. total volume of 0.5 ml and incubated for 30 – 60 min on vitronectin- Proteins and Antibodies—Native vitronectin was purified from coated plates. After incubation, plates were washed twice with PBS, pooled human plasma according to the procedure of Dahlback and and bound cells were enumerated using an ocular micrometer and Podack (8). Briefly, pooled plasma was subjected to salt fractionation counting six high-powered fields. The effect of RGDS, RGES, followed by ion exchange, dye affinity, and gel filtration chromatogra- proMMP-2, and active MMP-2 on adhesion to vitronectin was deter- phy. Presence and purity of vitronectin in each fraction was assessed by mined by addition of cells in the presence of various concentrations of both dot blot and electrophoretic analysis on 5–15% gradient SDS- peptide or proteinase to vitronectin-coated wells. polyacrylamide gels. ProMMP-2 and proMMP-2/TIMP-2 complex were Analysis of MMP-2 Membrane Association—To analyze binding of purified as described previously (9, 10). Plasminogen was purified from proMMP-2 or proMMP-2/TIMP-2 to intact melanoma cells, proteins human plasma by affinity chromatography on L-lysine-Sepharose and were labeled with I using Iodogen (Pierce) according to the manufac- separated into isoforms 1 and 2 as described previously (11). Plasmin turer’s specifications. Labeled protein (100 nM in minimal essential was generated by incubating 100 mg of plasminogen with 100 mlof medium containing 0.1% bovine serum albumin) was added to B16F10 urinary-plasminogen activator-Sepharose (Calbiochem) in 10 mM cells (5 3 10 )for1hat4 °Cinthe presence of increasing concentra- Hepes, pH 7.4, at 25 °C for 12 h followed by centrifugation to remove the tions of RGDS or RGES peptide (0 –1000 mg/ml). Cells were washed resin. Protein concentrations were determined spectrophotometrically 1% three times with Hanks’ balanced salt solution, and cell associated at 280 nm using an A value of 16.8 and molecular masses of 92 and 1cm radioactivity was measured in a g-counter. To assess the interaction of 81 kDa for plasminogen and plasmin, respectively. Endoproteinase proMMP-2 or proMMP-2/TIMP-2 with isolated melanoma cell mem- Glu-C from Staphylococcus aureus strain V8 (endoproteinase V8) and branes, melanoma cells were cultured overnight in serum-free medium, soybean trypsin inhibitor were purchased from Sigma and were coupled released from the flask with EDTA, and washed three times with PBS. to cyanogen bromide-activated Sepharose 4B (Sigma) according to the The plasma membrane fractions of B16F1 (2 3 10 ) and B16F10 (4 3 manufacturer’s specifications. The synthetic peptide Arg-Gly-Asp-Ser 10 ) cells were isolated using nitrogen cavitation (350 p.s.i., 20 min) and (RGDS), which blocks avb3 and avb5-mediated adhesion to vitronectin differential centrifugation as described previously (17). Cell lysates (12), and the noninhibitory control peptide Gly-Asp-Glu-Ser (RGES) were treated with soybean trypsin inhibitor (20 mg/ml), leupeptin (25 were purchased from Sigma. mg/ml), elastatinal (25 mg/ml), 3,4 dichloroisocoumarin (0.05 mM), and Analysis of MMP-2 and TIMP-2 Secretion—Latent MMPs in concen- trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64, 0.01 trated conditioned medium were activated by incubation at 37 °C for 60 mM) to prevent proteolysis of membrane proteins. B16F1 and B16F10 min in the presence of 1.0 mM amino-phenylmercuric acetate (APMA). plasma membranes were incubated (2 h, 37 °C) with purified proMMP- Zymographic analysis of secreted MMPs was performed using 9% SDS- 2/TIMP-2 or proMMP-2 (100 nM)in20mM Tris, 5 mM CaCl ,25mM polyacrylamide gels containing co-polymerized gelatin as described pre- sucrose, pH 7.4 (Tris-Ca-sucrose). Control samples contained buffer viously (13). Reverse zymography for detection of TIMPs was performed alone. The membranes were recovered by centrifugation (50,000 3 g for using 15% SDS-polyacrylamide gels containing co-polymerized gelatin 30 min), supernatants were discarded, and pellets were washed twice in and reagents purchased from University Technologies International Tris-Ca-sucrose. Membrane extracts were prepared by incubating ali- (Calgary, Alberta, Canada) according to the manufacturer’s specifica- quots of membrane fractions with 1% Triton X-100 at 4 °C for 16 h tions. Inactivation of the TIMP(s) in conditioned medium by reductive followed by clarification by centrifugation (50,000 3 g for 30 min) as carboxymethylation was carried out as described by Salvesen and Na- described previously (17). After resuspension in Tris-Ca-sucrose, ali- gase (14). Briefly, conditioned medium was treated with 2 mM dithio- quots of membranes were analyzed for bound activity by gelatin zymog- threitol for1hat37 °C followed by incubation with iodoacetamide (5 14 14 raphy and by C-gelatin degradation (17, 18). C-Gelatin was incu- mM) for 15 min at 37 °C. bated for 22 h at 37 °C with treated membrane aliquots in Tris-Ca- In Vitro Invasion Assays—In vitro invasive activity was assessed by sucrose containing 50 mM NaCl and 0.5 mM APMA. Parallel determining the ability of cells to invade a synthetic basement mem- experiments contained 1 mM o-phenanthroline. Reactions were stopped brane (Matrigel, Becton Dickinson, Bedford, MA). Polycarbonate filters by trichloroacetic acid precipitation, and soluble radioactivity was de- (8-mm pore size, Becton Dickinson) were coated with Matrigel (11 mg/ termined by scintillation counting. Gelatin degradation by membrane- filter) and placed in modified Boyden chambers. Cells (1 3 10 ) were associated proteinase was determined relative to membrane-free con- added to the top of the chamber in serum-free medium in the presence trols. The effect of membrane association on the gelatinolytic activity of or absence of vitronectin, plasmin-treated vitronectin, RGDS, or RGES. pre-activated MMP-2 was also determined as described previously (17). Following incubation for 8 –24 h, noninvading cells were removed from the top surface of the membrane filter with a cotton swab, and filters were removed and stained with Diff-Quik (Fisher). Cells on the lower RESULTS surface of the filter were enumerated using an ocular micrometer and It has been previously reported that treatment of A375M counting a minimum of six high-powered fields. In some experiments, 5 melanoma cells with antibodies directed against the vitronec- untreated cells (1 3 10 ) were preincubated for 90 min with MMP-2 (50 tin-binding integrin avb3 enhanced MMP-2 secretion and stim- nM) in serum-free culture medium containing 3% bovine serum albu- min. Cells were pelleted, washed twice with PBS to remove unbound ulated cellular invasion through Matrigel (7). To analyze the MMP-2, and enumerated, and invasiveness was assayed as described direct effect of vitronectin on melanoma MMP-2 secretion, above. B16F1 and B16F10 cells were incubated with increasing con- Limited Proteolysis of Vitronectin—Limited plasmin proteolysis of centrations of vitronectin, and secretion of gelatin-degrading vitronectin was carried out by incubating vitronectin and plasmin in a MMPs was analyzed by zymography. A dose-dependent in- 20:1 (w/w) vitronectin:plasmin ratio for various time periods from 10 crease in secretion of a gelatinolytic metalloproteinase was minto3hat37 °C. After incubation, plasmin was removed from the reaction by the addition of soybean trypsin inhibitor-Sepharose, fol- observed in the conditioned medium of vitronectin-treated lowed by centrifugation to remove the plasmin-soybean trypsin inhibi- B16F1 cells (Fig. 1) and B16F10 cells (Fig. 2A). The gelatino- tor-Sepharose complex. Complete removal of plasmin was confirmed by lytic enzyme was activated by treatment with APMA, inhibited addition of the synthetic plasmin substrate D-Val-Leu-Lys-p-nitroani- by the zinc chelator o-phenanthroline (data not shown), and lide (Sigma) and monitoring absorbance at 405 nm. Limited proteolysis co-migrated with authentic MMP-2, suggesting its identity as of vitronectin by endoproteinase V8 was performed by incubating MMP-2. MMP-2 secretion was not detectable by zymography in vitronectin (200 mg) with 200 ml of endoproteinase V8-Sepharose for various time periods from 30 min to 18 h at 25 °C, followed by centrif- untreated B16F1 or B16F10 cells (Fig. 1, lane 1, and Fig. 2, ugation to remove the proteinase resin. Cleavage of vitronectin by lane 1), but it was observed in cells treated with as little as 10 MMP-2 was assessed by incubating vitronectin with activated MMP-2 mg/ml vitronectin. Addition of vitronectin in the presence of in a 20:1 vitronectin:MMP-2 (w/w) ratio in 50 mM Tris-HCl, 0.2 M NaCl, RGDS peptide inhibited the vitronectin-induced MMP-2 secre- 10 mM CaCl , pH 7.6 at 37 °C for 18 h. Intact vitronectin and vitronectin tion, demonstrating that direct interaction of vitronectin with treated with plasmin, endoproteinase V8, or MMP-2 were analyzed by its cellular receptor is required for MMP-2 induction (Fig. 1). electrophoresis on 5–15% SDS-polyacrylamide gradient gels followed by staining with Coomassie Blue. Furthermore, vitronectin-induced MMP-2 secretion was inhib- Vitronectin Induction of Matrix Metalloproteinase-2 145 FIG.1. Zymogram depicting MMP-2 activity in B16F1-condi- tioned medium. B16F1 cells (1 3 10 ) were cultured for 18 h in serum-free medium containing increasing amounts of vitronectin (0 – 200 mg/ml), and conditioned media were analyzed for MMP activity by gelatin zymography on 9% SDS-polyacrylamide gels. The lane desig- nated 1001 contained conditioned medium from cells cultured in the presence of 100 mg/ml of vitronectin and 500 mg/ml of RGDS. FIG.3. Effect of vitronectin on Matrigel invasion. B16F1 or B16F10 cells (1 3 10 ) were added to 8-mm pore size polycarbonate filters coated with Matrigel basement membrane extract as described under “Experimental Procedures.” Following incubation for either 8 (B16F10) or 17 (B16F1) h, membranes were removed and stained, and the invading cells were enumerated. Open bars, control B16F1 (desig- nated F1(2)) or B16F10 (designated F10(2)); solid bars, B16F1 (desig- nated F1(1)) or B16F10 (designated F10(1)) in the presence of 100 mg/ml vitronectin. Hatched bar (designated F1(*)), B16F1 cells in the presence of 100 mg/ml V8-degraded vitronectin. Experiments were per- formed in triplicate, and error bars represent S.D. FIG.2. MMP and TIMP activity in B16F10-conditioned me- dium. B16F10 cells (1 3 10 ) were cultured for 18 h in serum-free medium containing 0 (lane 1), 100 (lane 2), 200 (lane 3), or 300 (lane 4) mg/ml vitronectin, and conditioned media were analyzed for MMP ac- tivity by gelatin zymography on 9% SDS-polyacrylamide gels (A) and for TIMP activity by reverse zymography on 15% SDS-polyacrylamide gels (B and C). Samples in C were subjected to reductive carboxymethyla- FIG.4. Limited proteolysis of vitronectin. Vitronectin (200 mg) tion to inactivate TIMPs as described under “Experimental Procedures” was subjected to limited proteolysis with plasmin (A and C) or endo- prior to reverse zymography. Lane S, MMP-2 standard. proteinase V8 (B and C) and incubated with B16F10 cells (1 3 10 )in serum-free medium (1 ml) for 18 h. Conditioned media were analyzed for MMP activity by gelatin zymography on 9% SDS-polyacrylamide ited by colchicine, demonstrating that vitronectin does not dis- gels. A, lane 1, cells only; lane 2, cells 1 intact vitronectin; lane 3, cells place MMP-2 from a common cell surface receptor such as avb3 1 plasmin-degraded vitronectin; lane 4, MMP-2 standard. B, lane 1, (data not shown). Analysis of TIMP-2 activity by reverse zy- cells only; lane 2, cells 1 intact vitronectin; lane 3, cells 1 endoprotein- mography revealed a metalloproteinase inhibitor that co-mi- ase V8-degraded vitronectin; lane 4, MMP-2 standard. Panel C, control showing limited proteolysis of vitronectin. Vitronectin was incubated grated with TIMP-2 (M 21,000) in the conditioned medium of with plasmin (37 °C for 30 min) (lane 2) or endoproteinase V8 (37 °C for untreated cells (Fig. 2B, lane 1), which was increased in 2h)(lane 4) as described under “Experimental Procedures,” and reac- vitronectin-treated cells (Fig. 2B, lanes 2– 4). Incubation of the tion products were analyzed by electrophoresis on 5–15% gradient conditioned medium samples with dithiothreitol and iodoacet- SDS-polyacrylamide gels and stained with Coomassie Blue. Lanes 1 and 3, intact vitronectin (20 mg); lane 2, plasmin-degraded vitronectin amide, a procedure shown to denature TIMP to a noninhibitory (20 mg); lane 4, endoproteinase V8-degraded vitronectin (20 mg). D, form (14), removed the TIMP-2 activity from the conditioned effect of MMP-2 on vitronectin. Vitronectin (20 mg) was incubated with medium (Fig. 2C). Vitronectin treatment also enhanced cell purified APMA-activated MMP-2 (1 mg) for 18 h at 37 °C. Reaction surface-associated MMP-2. B16F1 and B16F10 cells cultured products were analyzed by electrophoresis on 5–15% gradient gels and with vitronectin (50 mg) displayed a 17 and 20% increase in cell stained with Coomassie Blue. Lane 1, vitronectin; lane 2, vitronectin 1 MMP-2. surface MMP-2, respectively, as determined by cell surface enzyme-linked immunosorbent assay using an anti-MMP-2 an- tibody and an alkaline-phosphatase-conjugated secondary an- In addition to metalloproteinases, B16 melanoma cells also tibody (15). To assess the functional effect of MMP-2 induction, secrete the serine proteinase tissue-type plasminogen activa- the ability of melanoma cells to penetrate a synthetic basement tor, which converts the plasma zymogen plasminogen to the membrane (Matrigel) was analyzed. Both B16F1 and B16F10 active proteinase plasmin (13). Plasmin is a broad spectrum cells displayed enhanced in vitro invasive behavior when cul- serine proteinase that degrades numerous extracellular matrix tured in the presence of vitronectin (Fig. 3). proteins, including vitronectin (19). To determine the effect of 146 Vitronectin Induction of Matrix Metalloproteinase-2 TABLE I TABLE II Effect of RGDS on adhesion of B16F1 and B16F10 cells to vitronectin Effect of RGDS on binding of proMMP-2 and proMMP-2/TIMP-2 to B16F10 cells Increasing concentrations of RGDS were added to melanoma cells, and adhesion to vitronectin-coated wells was quantitated as described I-labeled proMMP-2 or proMMP-2/TIMP-2 (100 nM) was added to under “Experimental Procedures.” Results are expressed relative to wells containing 5 3 10 B16F10 cells in the presence of increasing wells containing no RGDS. concentrations of RGDS or RGES peptide. Bound ligand was deter- mined as described under “Experimental Procedures.” Results are ex- Inhibition pressed as % bound relative to control wells containing no added pep- RGDS tide (designated 100%). ProMMP-2 binding was analyzed in triplicate, B16F1 B16F10 whereas proMMP-2/TIMP-2 data are the average of duplicate experi- mg/ml % ments. 00 0 ProMMP-2/TIMP- 50 21 6 3.5 16 6 9.0 ProMMP-2 bound 2 bound [Peptide] 100 44 6 1.7 40 6 11.9 200 89 6 0.6 72 6 16.0 RGDS RGES RGDS RGES mg/ml % % 0 100.0 6 2.9 100.0 6 2.9 100.0 100.0 1 92.8 6 0.8 97.4 6 2.1 105.0 94.0 10 91.5 6 9.4 95.1 6 3.3 89.6 98.0 100 91.8 6 11.1 94.5 6 4.9 85.0 96.0 1000 94.5 6 6.2 91.5 6 0.5 90.6 96.0 TABLE III Effect of MMP-2 on melanoma cell adhesion to vitronectin B16F10 cells (1.5 3 10 ) were added to 24-well culture plates coated with vitronectin in the presence or absence of pro- or active MMP-2, as indicated. After incubation for 40 min at 37 °C, plates were washed with PBS and fixed, and bound cells were enumerated using an ocular micrometer. Experiments were performed in triplicate, and S.D. is indicated. Treatment Concentration Adhesion ng/ml No. of cells/field Control 70.6 6 17.5 ProMMP-2 350 84.2 6 8.2 700 76.0 6 9.4 Active MMP-2 350 72.4 6 25.0 700 65.8 6 8.3 FIG.5. Effect of RGD peptides on vitronectin-enhanced Matri- gel invasion. B16F1 cells (1 3 10 ) were added to 8-mm pore size polycarbonate filters coated with Matrigel in the presence (hatched binding of RGDS to vitronectin receptors on B16F1 and bars) or absence (open bars)of100 mg/ml vitronectin. RGDS or RGES B16F10 cells, as evidenced by the ability of the peptide to (100 mg/ml) was added to control or vitronectin-containing samples as inhibit melanoma cell adhesion to vitronectin in a concentration- indicated. Following incubation for 23 h, membranes were removed and stained, and invading cells were enumerated. Experiments were per- dependent manner (Table I). Incubation of either B16F1 or formed in triplicate, and error bars reflect S.D. B16F10 cells with up to 1 mg/ml of RGDS or RGES did not induce MMP-2 secretion (data not shown). However, simulta- plasmin-degraded vitronectin on melanoma cell MMP-2 secre- neous addition of vitronectin and RGDS inhibited vitronectin- tion, vitronectin was subjected to limited proteolysis with plas- induced MMP-2 secretion as shown in Fig. 1 (lane designated min (Fig. 4C, lane 2) prior to incubation with melanoma cells. 1001). Furthermore, inhibition of vitronectin-enhanced Matri- In contrast to results observed with intact vitronectin (Fig. 4A, gel invasion was 50% greater using RGDS peptide as compared lane 2), plasmin-degraded vitronectin did not induce MMP-2 with RGES (Fig. 5), further demonstrating that interaction of secretion (Fig. 4A, lane 3). Decreasing the extent of proteolysis the RGD sequence of vitronectin with a cellular integrin(s) is did not reinstate the stimulatory effect (data not shown). Be- necessary for MMP-2 induction. cause plasmin has trypsin-like specificity and therefore may Because recent data indicate that MMP activity may be cleave within the RGD site utilized by melanoma cells for regulated posttranslationally by interaction with the cell sur- integrin-mediated vitronectin binding, limited proteolysis of face (17, 22–27), the ability of MMP-2 to associate with the vitronectin with endoproteinase V8 (which has Glu specificity plasma membrane of B16F1 or B16F10 cells was determined. (20)) was also performed (Fig. 4C, lane 4). Similar to results Analysis of MMP-2 binding to the membranes of intact mela- obtained with plasmin, endoproteinase V8-cleaved vitronectin noma cells was assessed using I-labeled proMMP-2 or also failed to induce melanoma cell MMP-2 production (Fig. 4B, proMMP-2/TIMP-2. Attempts to determine an equilibrium dis- lane 3). Furthermore, Matrigel invasion was not stimulated by sociation constant for binding of either proMMP-2 or proMMP- endoproteinase V8-cleaved vitronectin (Fig. 3, hatched bar). To 2/TIMP-2 to intact cells were unsuccessful because saturation determine whether MMP-2 itself may initiate vitronectin deg- of binding sites was not achieved in the presence of I-labeled radation, vitronectin was incubated with APMA-activated ligand concentrations up to 500 nM (data not shown). These MMP-2 for 18 h at 37 °C at a 1:20 MMP-2:vitronectin ratio and data suggest that proMMP-2 and/or proMMP-2/TIMP-2 inter- analyzed by electrophoresis on a 5–15% SDS-polyacrylamide acts with a prevalent cell-associated protein such as collagen or gel. No change in the electrophoretic migration of MMP-2- fibronectin (25–26) or that binding is mediated by a receptor treated vitronectin was observed (Fig. 4D, lane 2), demonstrat- present in low abundance such that specific binding is obscured ing that vitronectin is not susceptible to proteolysis by MMP-2. by nonspecific interactions. To determine whether proMMP-2 To analyze further the effect of ligating the melanoma cell or proMMP-2/TIMP-2 may associate with vitronectin binding vitronectin receptor on MMP-2 secretion, the peptide RGDS, integrins, binding experiments were performed in the presence which interacts with both the avb3 and avb5 vitronectin re- of RGDS. No significant change in the amount of bound ceptors (12), was utilized. Control experiments demonstrated proMMP-2 or proMMP-2/TIMP-2 was observed in the presence Vitronectin Induction of Matrix Metalloproteinase-2 147 TABLE IV Effect of cell-associated MMP-2 on Matrigel invasion B16F1 cells were incubated for 90 min in serum-free medium with 3% BSA containing 50 nM proMMP-2 and washed twice with PBS to remove unbound proMMP-2, and cells (1 3 10 ) were added to 8 mm pore size polycarbonate filters coated with Matrigel. Following incubation for 17 h, membranes were removed and stained, and invading cells were enumerated. Sample Relative invasion No. of cells/field Control 22.0 6 5.9 150 nM proMMP-2 68.4 6 11.6 of up to 1 mg/ml (2.3 mM) RGDS or control peptide RGES (Table II), suggesting that interaction of proMMP-2 and proMMP-2/ TIMP-2 with intact murine melanoma cells is not mediated by vitronectin-binding integrins. This was confirmed by adhesion experiments that demonstrated that MMP-2, whether in proenzyme or active form, was unable to inhibit melanoma cell adhesion to vitronectin (Table III). In addition to binding to intact cells, exogenous proMMP-2 (data not shown) and proMMP-2/TIMP-2 bound to isolated plasma membranes from both B16F1 and B16F10 cells and retained hydrolytic activity against a macromolecular gelatin substrate (Fig. 6, A and B). Gelatinolytic activity was not detected in either clone of B16 plasma membranes prior to the addition of exogenous proMMP-2 or proMMP-2/TIMP-2 (Fig. 6), which is consistent with the lack of MMP-2 secretion ob- served in unstimulated cells (Figs. 1 and 2). The membrane- associated gelatinolytic activity was fully inhibited by addition of o-phenanthroline (Fig. 6B). Furthermore, membrane associ- ation enhanced the catalytic activity of activated MMP-2/ TIMP-2. Incubation of MMP-2/TIMP-2 with either plasma membranes or detergent soluble extracts from B16F1 and B16F10 cells resulted in a 33–76% increase in gelatinolytic activity (Fig. 6C), suggesting that membrane association may function as a mechanism for posttranslational regulation of MMP-2/TIMP-2 activity in melanoma cells. To assess the po- tential functional consequence of MMP-2 cellular association on invasive behavior, cells were incubated with 50 nM MMP-2 in serum-free culture medium containing 3% bovine serum albumin, washed to remove unbound MMP-2, and analyzed in a Matrigel invasion assay. A 3-fold increase in invasiveness was observed in cells containing bound MMP-2 relative to controls (Table IV), demonstrating that cellular association of MMP-2 contributes functionally to enhanced invasiveness. DISCUSSION The initial site of melanoma cell metastasis in vivo is fre- quently the regional lymph nodes, and the appearance of lymph node metastases is correlated with poor prognosis (3). Studies of experimental metastasis using B16F1 and B16F10 cells demonstrated similar incidence of lymph node metastases, al- though animals injected with B16F10 cells were more likely to develop pulmonary metastases (28). Expression of the vitronec- FIG.6. MMP-2 association with melanoma cell plasma mem- tin-binding integrin avb3 is enhanced in metastatic melanoma 8 8 branes. A, B16F1 (2 3 10 ) and B16F10 (4 3 10 ) cells were fraction- cells relative to parental nonmetastatic variants and recent ated as described under “Experimental Procedures,” and plasma mem- branes (P.M.) were incubated for2hat37 °Cinthe absence (control)or presence (1MMP-2) of purified proMMP-2/TIMP-2 (100 nM). Mem- represent S.D. C, effect of membrane association on MMP-2 catalytic branes were washed to remove unbound complex, treated with APMA, and analyzed by gelatin zymography to detect bound enzyme. B, plasma activity. C-gelatin cleavage by APMA-activated MMP-2/TIMP-2 (10 nM) was determined in the absence of membranes or in the presence of membranes (mem) from B16F1 or B16F10 cells were incubated in the presence (1) or absence (2) of proMMP-2/TIMP-2 as indicated, and B16 plasma membranes (1 mg) (open bars) or Triton X-100 detergent extracts (100 ng) (hatched bars). Data are presented as the percent bound enzyme was analyzed by incubating membrane aliquots with C-gelatin in Tris-Ca-sucrose containing 50 mM NaCl and 0.5 mM increase in gelatin hydrolysis relative to the activity of control MMP- 2/TIMP-2 determined in the absence of membranes. Experiments were APMA in the presence (solid bars) or absence (hatched bars)of1mM o-phenanthroline. Reactions were terminated by trichloroacetic acid performed in triplicate, and error bars represent S.D. In additional controls, the membrane preparations were incubated with the gelatin precipitation, and soluble radioactivity was determined by scintillation counting. Experiments were performed in quadruplicate, and error bars substrate in the absence of enzyme (data not shown). 148 Vitronectin Induction of Matrix Metalloproteinase-2 experiments have demonstrated that adhesion of metastatic B16F1 and B16F10 cells; however, limited proteolysis of melanoma cells to lymph node sections is blocked by either vitronectin removed the stimulatory effect, regardless of anti-avb3 or RGD-containing peptides (3). In related experi- whether the RGD site was disrupted (plasmin) or remained ments, ligation of melanoma cell avb3 using an anti-integrin intact (endoproteinase V8). Furthermore, simple ligation of the antibody was shown to enhance cellular invasiveness in vitro, vitronectin receptor with the peptide RGDS also failed to in- and conditioned medium from cells treated with anti-avb3 duce MMP-2 activity at concentrations well in excess of that displayed increased MMP-2 activity (7). This is particularly required for inhibition of cell adhesion. However, antibody li- gation of melanoma cell avb3, which can induce receptor ag- interesting in light of previous data that indicate that MMP-2 expression by melanoma cells correlates with increased inva- gregation, stimulated MMP-2 production (7). Together, these siveness, and it provides a biochemical mechanism whereby data indicate that a multivalent ligand-receptor interaction, invasion may be enhanced (29 –31). rather than simple ligand occupancy, is required for induction These observations are supported by data from the present of MMP-2. In light of these results, it is interesting to consider recent biophysical data that demonstrate that under physio- study that demonstrate a direct dose-dependent increase in MMP-2 secretion in vitronectin-treated B16F1 and B16F10 logic conditions, vitronectin can exist in both monomeric and melanoma cells. As a functional consequence of increased multimeric forms (39). Vitronectin in extravascular sites (i.e. matrix and tissue-associated) is predominantly in the multim- MMP-2 levels, cellular invasiveness is also enhanced. The vitronectin-induced increase in MMP-2 secretion and invasive eric form, suggesting that multivalent ligand-receptor interac- activity was abolished by RGDS peptide, providing evidence tions may prevail in vivo (39, 40). Furthermore, the current data suggest a biologic control mechanism whereby the stimu- that vitronectin interaction with cellular integrins regulates invasive behavior. Concomitant with MMP-2 secretion, levels lus for MMP-2 induction, i.e. intact vitronectin, may be re- moved. It is interesting to speculate that vitronectin-adherent of TIMP-2 were also increased by vitronectin treatment. Pre- melanoma cells, which also catalyze tissue-type plasminogen vious studies have shown that MMP-2 is secreted as a proen- zyme in complex with TIMP-2 by melanoma cells and other cell activator-mediated plasmin generation (13), may initiate plas- min-dependent proteolysis of vitronectin, thereby disrupting types, and additional reports suggest that the presence of the multivalent signal necessary for MMP-2 induction. TIMP-2 in this proenzyme-inhibitor complex is required for cellular activation of the MMP-2 zymogen by membrane-type Acknowledgments—We thank Dr. C. N. Rao (Department of Derma- MMPs (24, 32–36). tology, Northwestern University) for assistance with the in vitro inva- Recent evidence indicates that cell surface association may sion assay. function as a mechanism for posttranslational regulation of REFERENCES MMP activity (17, 21–26). The present data demonstrate that 1. Ruoslahti, E., and Giancotti, F. G. (1989) Cancer Cells 1, 119 –126 MMP-2/TIMP-2 associates with the plasma membrane fraction 2. Hynes, R. O. (1992) Cell 69, 11–25 of murine melanoma cells and exhibits enhanced catalytic ac- 3. Nip, J., Shibata, H. R., Loskutoff, D. J., Cheresh, D. A., and Brodt, P. (1992) tivity against macromolecular substrates relative to the solu- J. Clin. Invest. 90, 1406 –1413 4. Albelda, S. M., Mette, S. A., Elder, D. E., Stewart, R., Damjanovich, L., Herlyn, tion phase enzyme supporting the role of membrane associa- M., and Buck, C. A. (1990) Cancer Res. 50, 6757– 6764 tion in MMP regulation. The mechanism of interaction of 5. Sanders, L. 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R., Huang, W., and 265, 18490 –18498 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Intact Vitronectin Induces Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinases-2 Expression and Enhanced Cellular Invasion by Melanoma Cells

Bafetti, Lisa M.; Young, Timothy N.; Itoh, Yoshifumi; Stack, M. Sharon
Journal of Biological Chemistry – Jan 1, 1998
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 1, Issue of January 2, pp. 143–149, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Intact Vitronectin Induces Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinases-2 Expression and Enhanced Cellular Invasion by Melanoma Cells* (Received for publication, October 11, 1996, and in revised form, September 25, 1997) Lisa M. Bafetti‡, Timothy N. Young§, Yoshifumi Itoh‡, and M. Sharon Stack‡¶i From the Departments of ‡Obstetrics & Gynecology and ¶Cell & Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611 and §Washington University School of Medicine, St. Louis, Missouri 63110 The initial site of melanoma cell metastasis is fre- adhesion to the regional lymph nodes, which correlates with poor prognosis, is mediated via interaction of specific integrins quently the regional lymph nodes, and the appearance of lymph node metastasis correlates with poor progno- on the melanoma cell surface with lymph node vitronectin (3). sis. Lymph node adhesion is mediated by an interaction Previous data have demonstrated a relationship between ele- avb3 and lymph node between the tumor cell integrin vated levels of vitronectin-binding integrins and increased mel- vitronectin. In this study, we explored the relationship anoma cell invasiveness (4 – 6). Furthermore, ligation of the between adhesion and proteolysis by examining the di- avb3 integrin on melanoma cells by anti-avb3 antibodies en- rect effect of vitronectin receptor ligation on matrix hances secretion of matrix metalloproteinase-2 (MMP-2, gela- metalloproteinase-2 (MMP-2) production by B16F1 and tinase A, 72-kDa type IV collagenase), resulting in increased B16F10 melanoma cells. We report a dose-dependent in- cellular invasiveness (7). Together, these data suggest that inte- crease in secretion of both MMP-2 and tissue inhibitor of grin-mediated binding of tumor cells to a specific matrix-associ- metalloproteinases-2 (TIMP-2) in response to vitronec- ated protein, such as vitronectin, can promote tumor cell inva- tin. Cellular invasiveness was also enhanced by sion by increasing the levels of a matrix-degrading proteinase. vitronectin, as shown by the increased ability of The majority of integrins recognize multiple extracellular vitronectin-treated cells to invade a synthetic basement matrix ligands (1, 2), and precise biologic responses may be membrane (Matrigel). Both the vitronectin-induced regulated by differential integrin ligation with distinct extra- MMP-2 production and vitronectin-enhanced invasion cellular matrix proteins. In this study, we have explored the were blocked by the peptide ligand Arg-Gly-Asp-Ser direct effect of the matrix-associated ligand vitronectin on pro- (RGDS). Furthermore, neither plasmin-degraded duction of MMP-2 by melanoma cells. We report a dose-depend- vitronectin nor the peptide ligand RGDS stimulated ent increase in secretion of both MMP-2 and tissue inhibitor of MMP-2 secretion or invasiveness, indicating that a mul- metalloproteinases-2 (TIMP-2), as well as enhanced cellular tivalent ligand-receptor interaction rather than simple invasiveness, in response to vitronectin. Intact vitronectin is receptor occupancy was required for MMP-2 induction. MMP-2 and MMP-2/TIMP-2 interaction with the plasma required for MMP-2 induction because neither plasmin-treated membrane of melanoma cells resulted in enhanced cat- vitronectin nor a peptide ligand (Arg-Gly-Asp-Ser (RGDS)) al- C-labeled gelatin, suggesting alytic activity against ters MMP-2 secretion, indicating the requirement for a multi- that membrane association may function in posttrans- valent ligand-receptor interaction. Furthermore, MMP-2 inter- lational regulation of MMP-2 activity. This is supported acts with the plasma membranes of melanoma cells, exhibits by data showing increased cellular invasion by cells enhanced catalytic activity relative to the solution phase en- containing membrane-bound MMP-2. Binding of zyme, and increases cellular invasive activity. Membrane bind- proMMP-2 and proMMP-2/TIMP-2 to melanoma cells ing of MMP-2 is unaffected by RGDS, and melanoma cell ad- was not inhibited by RGDS, and melanoma cell adhesion hesion to vitronectin is not inhibited by MMP-2, indicating that to vitronectin was unaffected by pro- or active MMP-2, MMP-2 does not bind the vitronectin receptor on murine mel- indicating that MMP-2 did not interact with the murine anoma cells. These data suggest a potential physiologic mech- vitronectin receptor. Together, these data provide evi- anism whereby the relative integrity of the adhesive substra- dence for a functional link between adhesion and pro- tum may differentially regulate secretion and activity of a teolysis and suggest a potential mechanism whereby matrix-degrading proteinase and subsequent cellular invasive adhesion of an invasive cell to the extracellular matrix behavior. regulates subsequent invasive behavior. EXPERIMENTAL PROCEDURES Cell Culture—The murine B16F10 and B16F1 melanoma cell lines Adhesion of tumor cells to specific extracellular matrix mac- were obtained from Dr. I. J. Fidler (The University of Texas M. D. romolecules is an initial component of the metastatic process Anderson Hospital) and were cultured in Eagle’s minimal essential (reviewed in Refs. 1 and 2). In metastatic melanoma, tumor cell medium supplemented with 5% fetal calf serum, nonessential amino acids, L-glutamine, and vitamins. Prior to each experiment, cells were washed with calcium and magnesium-free Dulbecco’s phosphate-buff- * This work was supported by Research Grant CA 58900 from the ered saline (PBS) and incubated for 2 min at 25 °C with 1 mM EDTA in NCI, National Institutes of Health (to M. S. S.). The costs of publication serum-free Eagle’s minimal essential medium to release cells from the of this article were defrayed in part by the payment of page charges. culture flask. Cells were seeded in 1 ml of serum-free Eagle’s minimal This article must therefore be hereby marked “advertisement”inac- essential medium at a density of 1 3 10 cells/ml and incubated 18 h at cordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: Northwestern Uni- versity Medical School, 303 East Chicago Ave., Tarry 4-751, Chicago, The abbreviations used are: MMP-2, matrix metalloproteinases-2; IL 60611. Tel.: 312-908-8216; Fax: 312-908-8773; E-mail: mss130@ TIMP, tissue inhibitor of metalloproteinases; PBS, phosphate-buffered anima.nums.nwu.edu. saline; APMA, amino-phenylmercuric acetate. This paper is available on line at http://www.jbc.org 143 This is an Open Access article under the CC BY license. 144 Vitronectin Induction of Matrix Metalloproteinase-2 37 °C in the presence of vitronectin, peptide, or both as described below. Adhesion Assay—Vitronectin was passively adsorbed to 24-well cul- ture plates as described previously (15), blocked by incubating with In some experiments, colchicine (0.2 mM) was included for inhibition of PBS containing 2% bovine serum albumin for2hat25°C,and utilized secretion. Conditioned media were removed and concentrated 4-fold at for cell attachment assays as described by Pierschbacher and Ruoslahti 4 °C using a Centricon 10 (Amicon) concentrator and analyzed imme- (16). Briefly, cells were plated at a constant of 1.5 3 10 cells/ml in a diately as described below. total volume of 0.5 ml and incubated for 30 – 60 min on vitronectin- Proteins and Antibodies—Native vitronectin was purified from coated plates. After incubation, plates were washed twice with PBS, pooled human plasma according to the procedure of Dahlback and and bound cells were enumerated using an ocular micrometer and Podack (8). Briefly, pooled plasma was subjected to salt fractionation counting six high-powered fields. The effect of RGDS, RGES, followed by ion exchange, dye affinity, and gel filtration chromatogra- proMMP-2, and active MMP-2 on adhesion to vitronectin was deter- phy. Presence and purity of vitronectin in each fraction was assessed by mined by addition of cells in the presence of various concentrations of both dot blot and electrophoretic analysis on 5–15% gradient SDS- peptide or proteinase to vitronectin-coated wells. polyacrylamide gels. ProMMP-2 and proMMP-2/TIMP-2 complex were Analysis of MMP-2 Membrane Association—To analyze binding of purified as described previously (9, 10). Plasminogen was purified from proMMP-2 or proMMP-2/TIMP-2 to intact melanoma cells, proteins human plasma by affinity chromatography on L-lysine-Sepharose and were labeled with I using Iodogen (Pierce) according to the manufac- separated into isoforms 1 and 2 as described previously (11). Plasmin turer’s specifications. Labeled protein (100 nM in minimal essential was generated by incubating 100 mg of plasminogen with 100 mlof medium containing 0.1% bovine serum albumin) was added to B16F10 urinary-plasminogen activator-Sepharose (Calbiochem) in 10 mM cells (5 3 10 )for1hat4 °Cinthe presence of increasing concentra- Hepes, pH 7.4, at 25 °C for 12 h followed by centrifugation to remove the tions of RGDS or RGES peptide (0 –1000 mg/ml). Cells were washed resin. Protein concentrations were determined spectrophotometrically 1% three times with Hanks’ balanced salt solution, and cell associated at 280 nm using an A value of 16.8 and molecular masses of 92 and 1cm radioactivity was measured in a g-counter. To assess the interaction of 81 kDa for plasminogen and plasmin, respectively. Endoproteinase proMMP-2 or proMMP-2/TIMP-2 with isolated melanoma cell mem- Glu-C from Staphylococcus aureus strain V8 (endoproteinase V8) and branes, melanoma cells were cultured overnight in serum-free medium, soybean trypsin inhibitor were purchased from Sigma and were coupled released from the flask with EDTA, and washed three times with PBS. to cyanogen bromide-activated Sepharose 4B (Sigma) according to the The plasma membrane fractions of B16F1 (2 3 10 ) and B16F10 (4 3 manufacturer’s specifications. The synthetic peptide Arg-Gly-Asp-Ser 10 ) cells were isolated using nitrogen cavitation (350 p.s.i., 20 min) and (RGDS), which blocks avb3 and avb5-mediated adhesion to vitronectin differential centrifugation as described previously (17). Cell lysates (12), and the noninhibitory control peptide Gly-Asp-Glu-Ser (RGES) were treated with soybean trypsin inhibitor (20 mg/ml), leupeptin (25 were purchased from Sigma. mg/ml), elastatinal (25 mg/ml), 3,4 dichloroisocoumarin (0.05 mM), and Analysis of MMP-2 and TIMP-2 Secretion—Latent MMPs in concen- trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64, 0.01 trated conditioned medium were activated by incubation at 37 °C for 60 mM) to prevent proteolysis of membrane proteins. B16F1 and B16F10 min in the presence of 1.0 mM amino-phenylmercuric acetate (APMA). plasma membranes were incubated (2 h, 37 °C) with purified proMMP- Zymographic analysis of secreted MMPs was performed using 9% SDS- 2/TIMP-2 or proMMP-2 (100 nM)in20mM Tris, 5 mM CaCl ,25mM polyacrylamide gels containing co-polymerized gelatin as described pre- sucrose, pH 7.4 (Tris-Ca-sucrose). Control samples contained buffer viously (13). Reverse zymography for detection of TIMPs was performed alone. The membranes were recovered by centrifugation (50,000 3 g for using 15% SDS-polyacrylamide gels containing co-polymerized gelatin 30 min), supernatants were discarded, and pellets were washed twice in and reagents purchased from University Technologies International Tris-Ca-sucrose. Membrane extracts were prepared by incubating ali- (Calgary, Alberta, Canada) according to the manufacturer’s specifica- quots of membrane fractions with 1% Triton X-100 at 4 °C for 16 h tions. Inactivation of the TIMP(s) in conditioned medium by reductive followed by clarification by centrifugation (50,000 3 g for 30 min) as carboxymethylation was carried out as described by Salvesen and Na- described previously (17). After resuspension in Tris-Ca-sucrose, ali- gase (14). Briefly, conditioned medium was treated with 2 mM dithio- quots of membranes were analyzed for bound activity by gelatin zymog- threitol for1hat37 °C followed by incubation with iodoacetamide (5 14 14 raphy and by C-gelatin degradation (17, 18). C-Gelatin was incu- mM) for 15 min at 37 °C. bated for 22 h at 37 °C with treated membrane aliquots in Tris-Ca- In Vitro Invasion Assays—In vitro invasive activity was assessed by sucrose containing 50 mM NaCl and 0.5 mM APMA. Parallel determining the ability of cells to invade a synthetic basement mem- experiments contained 1 mM o-phenanthroline. Reactions were stopped brane (Matrigel, Becton Dickinson, Bedford, MA). Polycarbonate filters by trichloroacetic acid precipitation, and soluble radioactivity was de- (8-mm pore size, Becton Dickinson) were coated with Matrigel (11 mg/ termined by scintillation counting. Gelatin degradation by membrane- filter) and placed in modified Boyden chambers. Cells (1 3 10 ) were associated proteinase was determined relative to membrane-free con- added to the top of the chamber in serum-free medium in the presence trols. The effect of membrane association on the gelatinolytic activity of or absence of vitronectin, plasmin-treated vitronectin, RGDS, or RGES. pre-activated MMP-2 was also determined as described previously (17). Following incubation for 8 –24 h, noninvading cells were removed from the top surface of the membrane filter with a cotton swab, and filters were removed and stained with Diff-Quik (Fisher). Cells on the lower RESULTS surface of the filter were enumerated using an ocular micrometer and It has been previously reported that treatment of A375M counting a minimum of six high-powered fields. In some experiments, 5 melanoma cells with antibodies directed against the vitronec- untreated cells (1 3 10 ) were preincubated for 90 min with MMP-2 (50 tin-binding integrin avb3 enhanced MMP-2 secretion and stim- nM) in serum-free culture medium containing 3% bovine serum albu- min. Cells were pelleted, washed twice with PBS to remove unbound ulated cellular invasion through Matrigel (7). To analyze the MMP-2, and enumerated, and invasiveness was assayed as described direct effect of vitronectin on melanoma MMP-2 secretion, above. B16F1 and B16F10 cells were incubated with increasing con- Limited Proteolysis of Vitronectin—Limited plasmin proteolysis of centrations of vitronectin, and secretion of gelatin-degrading vitronectin was carried out by incubating vitronectin and plasmin in a MMPs was analyzed by zymography. A dose-dependent in- 20:1 (w/w) vitronectin:plasmin ratio for various time periods from 10 crease in secretion of a gelatinolytic metalloproteinase was minto3hat37 °C. After incubation, plasmin was removed from the reaction by the addition of soybean trypsin inhibitor-Sepharose, fol- observed in the conditioned medium of vitronectin-treated lowed by centrifugation to remove the plasmin-soybean trypsin inhibi- B16F1 cells (Fig. 1) and B16F10 cells (Fig. 2A). The gelatino- tor-Sepharose complex. Complete removal of plasmin was confirmed by lytic enzyme was activated by treatment with APMA, inhibited addition of the synthetic plasmin substrate D-Val-Leu-Lys-p-nitroani- by the zinc chelator o-phenanthroline (data not shown), and lide (Sigma) and monitoring absorbance at 405 nm. Limited proteolysis co-migrated with authentic MMP-2, suggesting its identity as of vitronectin by endoproteinase V8 was performed by incubating MMP-2. MMP-2 secretion was not detectable by zymography in vitronectin (200 mg) with 200 ml of endoproteinase V8-Sepharose for various time periods from 30 min to 18 h at 25 °C, followed by centrif- untreated B16F1 or B16F10 cells (Fig. 1, lane 1, and Fig. 2, ugation to remove the proteinase resin. Cleavage of vitronectin by lane 1), but it was observed in cells treated with as little as 10 MMP-2 was assessed by incubating vitronectin with activated MMP-2 mg/ml vitronectin. Addition of vitronectin in the presence of in a 20:1 vitronectin:MMP-2 (w/w) ratio in 50 mM Tris-HCl, 0.2 M NaCl, RGDS peptide inhibited the vitronectin-induced MMP-2 secre- 10 mM CaCl , pH 7.6 at 37 °C for 18 h. Intact vitronectin and vitronectin tion, demonstrating that direct interaction of vitronectin with treated with plasmin, endoproteinase V8, or MMP-2 were analyzed by its cellular receptor is required for MMP-2 induction (Fig. 1). electrophoresis on 5–15% SDS-polyacrylamide gradient gels followed by staining with Coomassie Blue. Furthermore, vitronectin-induced MMP-2 secretion was inhib- Vitronectin Induction of Matrix Metalloproteinase-2 145 FIG.1. Zymogram depicting MMP-2 activity in B16F1-condi- tioned medium. B16F1 cells (1 3 10 ) were cultured for 18 h in serum-free medium containing increasing amounts of vitronectin (0 – 200 mg/ml), and conditioned media were analyzed for MMP activity by gelatin zymography on 9% SDS-polyacrylamide gels. The lane desig- nated 1001 contained conditioned medium from cells cultured in the presence of 100 mg/ml of vitronectin and 500 mg/ml of RGDS. FIG.3. Effect of vitronectin on Matrigel invasion. B16F1 or B16F10 cells (1 3 10 ) were added to 8-mm pore size polycarbonate filters coated with Matrigel basement membrane extract as described under “Experimental Procedures.” Following incubation for either 8 (B16F10) or 17 (B16F1) h, membranes were removed and stained, and the invading cells were enumerated. Open bars, control B16F1 (desig- nated F1(2)) or B16F10 (designated F10(2)); solid bars, B16F1 (desig- nated F1(1)) or B16F10 (designated F10(1)) in the presence of 100 mg/ml vitronectin. Hatched bar (designated F1(*)), B16F1 cells in the presence of 100 mg/ml V8-degraded vitronectin. Experiments were per- formed in triplicate, and error bars represent S.D. FIG.2. MMP and TIMP activity in B16F10-conditioned me- dium. B16F10 cells (1 3 10 ) were cultured for 18 h in serum-free medium containing 0 (lane 1), 100 (lane 2), 200 (lane 3), or 300 (lane 4) mg/ml vitronectin, and conditioned media were analyzed for MMP ac- tivity by gelatin zymography on 9% SDS-polyacrylamide gels (A) and for TIMP activity by reverse zymography on 15% SDS-polyacrylamide gels (B and C). Samples in C were subjected to reductive carboxymethyla- FIG.4. Limited proteolysis of vitronectin. Vitronectin (200 mg) tion to inactivate TIMPs as described under “Experimental Procedures” was subjected to limited proteolysis with plasmin (A and C) or endo- prior to reverse zymography. Lane S, MMP-2 standard. proteinase V8 (B and C) and incubated with B16F10 cells (1 3 10 )in serum-free medium (1 ml) for 18 h. Conditioned media were analyzed for MMP activity by gelatin zymography on 9% SDS-polyacrylamide ited by colchicine, demonstrating that vitronectin does not dis- gels. A, lane 1, cells only; lane 2, cells 1 intact vitronectin; lane 3, cells place MMP-2 from a common cell surface receptor such as avb3 1 plasmin-degraded vitronectin; lane 4, MMP-2 standard. B, lane 1, (data not shown). Analysis of TIMP-2 activity by reverse zy- cells only; lane 2, cells 1 intact vitronectin; lane 3, cells 1 endoprotein- mography revealed a metalloproteinase inhibitor that co-mi- ase V8-degraded vitronectin; lane 4, MMP-2 standard. Panel C, control showing limited proteolysis of vitronectin. Vitronectin was incubated grated with TIMP-2 (M 21,000) in the conditioned medium of with plasmin (37 °C for 30 min) (lane 2) or endoproteinase V8 (37 °C for untreated cells (Fig. 2B, lane 1), which was increased in 2h)(lane 4) as described under “Experimental Procedures,” and reac- vitronectin-treated cells (Fig. 2B, lanes 2– 4). Incubation of the tion products were analyzed by electrophoresis on 5–15% gradient conditioned medium samples with dithiothreitol and iodoacet- SDS-polyacrylamide gels and stained with Coomassie Blue. Lanes 1 and 3, intact vitronectin (20 mg); lane 2, plasmin-degraded vitronectin amide, a procedure shown to denature TIMP to a noninhibitory (20 mg); lane 4, endoproteinase V8-degraded vitronectin (20 mg). D, form (14), removed the TIMP-2 activity from the conditioned effect of MMP-2 on vitronectin. Vitronectin (20 mg) was incubated with medium (Fig. 2C). Vitronectin treatment also enhanced cell purified APMA-activated MMP-2 (1 mg) for 18 h at 37 °C. Reaction surface-associated MMP-2. B16F1 and B16F10 cells cultured products were analyzed by electrophoresis on 5–15% gradient gels and with vitronectin (50 mg) displayed a 17 and 20% increase in cell stained with Coomassie Blue. Lane 1, vitronectin; lane 2, vitronectin 1 MMP-2. surface MMP-2, respectively, as determined by cell surface enzyme-linked immunosorbent assay using an anti-MMP-2 an- tibody and an alkaline-phosphatase-conjugated secondary an- In addition to metalloproteinases, B16 melanoma cells also tibody (15). To assess the functional effect of MMP-2 induction, secrete the serine proteinase tissue-type plasminogen activa- the ability of melanoma cells to penetrate a synthetic basement tor, which converts the plasma zymogen plasminogen to the membrane (Matrigel) was analyzed. Both B16F1 and B16F10 active proteinase plasmin (13). Plasmin is a broad spectrum cells displayed enhanced in vitro invasive behavior when cul- serine proteinase that degrades numerous extracellular matrix tured in the presence of vitronectin (Fig. 3). proteins, including vitronectin (19). To determine the effect of 146 Vitronectin Induction of Matrix Metalloproteinase-2 TABLE I TABLE II Effect of RGDS on adhesion of B16F1 and B16F10 cells to vitronectin Effect of RGDS on binding of proMMP-2 and proMMP-2/TIMP-2 to B16F10 cells Increasing concentrations of RGDS were added to melanoma cells, and adhesion to vitronectin-coated wells was quantitated as described I-labeled proMMP-2 or proMMP-2/TIMP-2 (100 nM) was added to under “Experimental Procedures.” Results are expressed relative to wells containing 5 3 10 B16F10 cells in the presence of increasing wells containing no RGDS. concentrations of RGDS or RGES peptide. Bound ligand was deter- mined as described under “Experimental Procedures.” Results are ex- Inhibition pressed as % bound relative to control wells containing no added pep- RGDS tide (designated 100%). ProMMP-2 binding was analyzed in triplicate, B16F1 B16F10 whereas proMMP-2/TIMP-2 data are the average of duplicate experi- mg/ml % ments. 00 0 ProMMP-2/TIMP- 50 21 6 3.5 16 6 9.0 ProMMP-2 bound 2 bound [Peptide] 100 44 6 1.7 40 6 11.9 200 89 6 0.6 72 6 16.0 RGDS RGES RGDS RGES mg/ml % % 0 100.0 6 2.9 100.0 6 2.9 100.0 100.0 1 92.8 6 0.8 97.4 6 2.1 105.0 94.0 10 91.5 6 9.4 95.1 6 3.3 89.6 98.0 100 91.8 6 11.1 94.5 6 4.9 85.0 96.0 1000 94.5 6 6.2 91.5 6 0.5 90.6 96.0 TABLE III Effect of MMP-2 on melanoma cell adhesion to vitronectin B16F10 cells (1.5 3 10 ) were added to 24-well culture plates coated with vitronectin in the presence or absence of pro- or active MMP-2, as indicated. After incubation for 40 min at 37 °C, plates were washed with PBS and fixed, and bound cells were enumerated using an ocular micrometer. Experiments were performed in triplicate, and S.D. is indicated. Treatment Concentration Adhesion ng/ml No. of cells/field Control 70.6 6 17.5 ProMMP-2 350 84.2 6 8.2 700 76.0 6 9.4 Active MMP-2 350 72.4 6 25.0 700 65.8 6 8.3 FIG.5. Effect of RGD peptides on vitronectin-enhanced Matri- gel invasion. B16F1 cells (1 3 10 ) were added to 8-mm pore size polycarbonate filters coated with Matrigel in the presence (hatched binding of RGDS to vitronectin receptors on B16F1 and bars) or absence (open bars)of100 mg/ml vitronectin. RGDS or RGES B16F10 cells, as evidenced by the ability of the peptide to (100 mg/ml) was added to control or vitronectin-containing samples as inhibit melanoma cell adhesion to vitronectin in a concentration- indicated. Following incubation for 23 h, membranes were removed and stained, and invading cells were enumerated. Experiments were per- dependent manner (Table I). Incubation of either B16F1 or formed in triplicate, and error bars reflect S.D. B16F10 cells with up to 1 mg/ml of RGDS or RGES did not induce MMP-2 secretion (data not shown). However, simulta- plasmin-degraded vitronectin on melanoma cell MMP-2 secre- neous addition of vitronectin and RGDS inhibited vitronectin- tion, vitronectin was subjected to limited proteolysis with plas- induced MMP-2 secretion as shown in Fig. 1 (lane designated min (Fig. 4C, lane 2) prior to incubation with melanoma cells. 1001). Furthermore, inhibition of vitronectin-enhanced Matri- In contrast to results observed with intact vitronectin (Fig. 4A, gel invasion was 50% greater using RGDS peptide as compared lane 2), plasmin-degraded vitronectin did not induce MMP-2 with RGES (Fig. 5), further demonstrating that interaction of secretion (Fig. 4A, lane 3). Decreasing the extent of proteolysis the RGD sequence of vitronectin with a cellular integrin(s) is did not reinstate the stimulatory effect (data not shown). Be- necessary for MMP-2 induction. cause plasmin has trypsin-like specificity and therefore may Because recent data indicate that MMP activity may be cleave within the RGD site utilized by melanoma cells for regulated posttranslationally by interaction with the cell sur- integrin-mediated vitronectin binding, limited proteolysis of face (17, 22–27), the ability of MMP-2 to associate with the vitronectin with endoproteinase V8 (which has Glu specificity plasma membrane of B16F1 or B16F10 cells was determined. (20)) was also performed (Fig. 4C, lane 4). Similar to results Analysis of MMP-2 binding to the membranes of intact mela- obtained with plasmin, endoproteinase V8-cleaved vitronectin noma cells was assessed using I-labeled proMMP-2 or also failed to induce melanoma cell MMP-2 production (Fig. 4B, proMMP-2/TIMP-2. Attempts to determine an equilibrium dis- lane 3). Furthermore, Matrigel invasion was not stimulated by sociation constant for binding of either proMMP-2 or proMMP- endoproteinase V8-cleaved vitronectin (Fig. 3, hatched bar). To 2/TIMP-2 to intact cells were unsuccessful because saturation determine whether MMP-2 itself may initiate vitronectin deg- of binding sites was not achieved in the presence of I-labeled radation, vitronectin was incubated with APMA-activated ligand concentrations up to 500 nM (data not shown). These MMP-2 for 18 h at 37 °C at a 1:20 MMP-2:vitronectin ratio and data suggest that proMMP-2 and/or proMMP-2/TIMP-2 inter- analyzed by electrophoresis on a 5–15% SDS-polyacrylamide acts with a prevalent cell-associated protein such as collagen or gel. No change in the electrophoretic migration of MMP-2- fibronectin (25–26) or that binding is mediated by a receptor treated vitronectin was observed (Fig. 4D, lane 2), demonstrat- present in low abundance such that specific binding is obscured ing that vitronectin is not susceptible to proteolysis by MMP-2. by nonspecific interactions. To determine whether proMMP-2 To analyze further the effect of ligating the melanoma cell or proMMP-2/TIMP-2 may associate with vitronectin binding vitronectin receptor on MMP-2 secretion, the peptide RGDS, integrins, binding experiments were performed in the presence which interacts with both the avb3 and avb5 vitronectin re- of RGDS. No significant change in the amount of bound ceptors (12), was utilized. Control experiments demonstrated proMMP-2 or proMMP-2/TIMP-2 was observed in the presence Vitronectin Induction of Matrix Metalloproteinase-2 147 TABLE IV Effect of cell-associated MMP-2 on Matrigel invasion B16F1 cells were incubated for 90 min in serum-free medium with 3% BSA containing 50 nM proMMP-2 and washed twice with PBS to remove unbound proMMP-2, and cells (1 3 10 ) were added to 8 mm pore size polycarbonate filters coated with Matrigel. Following incubation for 17 h, membranes were removed and stained, and invading cells were enumerated. Sample Relative invasion No. of cells/field Control 22.0 6 5.9 150 nM proMMP-2 68.4 6 11.6 of up to 1 mg/ml (2.3 mM) RGDS or control peptide RGES (Table II), suggesting that interaction of proMMP-2 and proMMP-2/ TIMP-2 with intact murine melanoma cells is not mediated by vitronectin-binding integrins. This was confirmed by adhesion experiments that demonstrated that MMP-2, whether in proenzyme or active form, was unable to inhibit melanoma cell adhesion to vitronectin (Table III). In addition to binding to intact cells, exogenous proMMP-2 (data not shown) and proMMP-2/TIMP-2 bound to isolated plasma membranes from both B16F1 and B16F10 cells and retained hydrolytic activity against a macromolecular gelatin substrate (Fig. 6, A and B). Gelatinolytic activity was not detected in either clone of B16 plasma membranes prior to the addition of exogenous proMMP-2 or proMMP-2/TIMP-2 (Fig. 6), which is consistent with the lack of MMP-2 secretion ob- served in unstimulated cells (Figs. 1 and 2). The membrane- associated gelatinolytic activity was fully inhibited by addition of o-phenanthroline (Fig. 6B). Furthermore, membrane associ- ation enhanced the catalytic activity of activated MMP-2/ TIMP-2. Incubation of MMP-2/TIMP-2 with either plasma membranes or detergent soluble extracts from B16F1 and B16F10 cells resulted in a 33–76% increase in gelatinolytic activity (Fig. 6C), suggesting that membrane association may function as a mechanism for posttranslational regulation of MMP-2/TIMP-2 activity in melanoma cells. To assess the po- tential functional consequence of MMP-2 cellular association on invasive behavior, cells were incubated with 50 nM MMP-2 in serum-free culture medium containing 3% bovine serum albumin, washed to remove unbound MMP-2, and analyzed in a Matrigel invasion assay. A 3-fold increase in invasiveness was observed in cells containing bound MMP-2 relative to controls (Table IV), demonstrating that cellular association of MMP-2 contributes functionally to enhanced invasiveness. DISCUSSION The initial site of melanoma cell metastasis in vivo is fre- quently the regional lymph nodes, and the appearance of lymph node metastases is correlated with poor prognosis (3). Studies of experimental metastasis using B16F1 and B16F10 cells demonstrated similar incidence of lymph node metastases, al- though animals injected with B16F10 cells were more likely to develop pulmonary metastases (28). Expression of the vitronec- FIG.6. MMP-2 association with melanoma cell plasma mem- tin-binding integrin avb3 is enhanced in metastatic melanoma 8 8 branes. A, B16F1 (2 3 10 ) and B16F10 (4 3 10 ) cells were fraction- cells relative to parental nonmetastatic variants and recent ated as described under “Experimental Procedures,” and plasma mem- branes (P.M.) were incubated for2hat37 °Cinthe absence (control)or presence (1MMP-2) of purified proMMP-2/TIMP-2 (100 nM). Mem- represent S.D. C, effect of membrane association on MMP-2 catalytic branes were washed to remove unbound complex, treated with APMA, and analyzed by gelatin zymography to detect bound enzyme. B, plasma activity. C-gelatin cleavage by APMA-activated MMP-2/TIMP-2 (10 nM) was determined in the absence of membranes or in the presence of membranes (mem) from B16F1 or B16F10 cells were incubated in the presence (1) or absence (2) of proMMP-2/TIMP-2 as indicated, and B16 plasma membranes (1 mg) (open bars) or Triton X-100 detergent extracts (100 ng) (hatched bars). Data are presented as the percent bound enzyme was analyzed by incubating membrane aliquots with C-gelatin in Tris-Ca-sucrose containing 50 mM NaCl and 0.5 mM increase in gelatin hydrolysis relative to the activity of control MMP- 2/TIMP-2 determined in the absence of membranes. Experiments were APMA in the presence (solid bars) or absence (hatched bars)of1mM o-phenanthroline. Reactions were terminated by trichloroacetic acid performed in triplicate, and error bars represent S.D. In additional controls, the membrane preparations were incubated with the gelatin precipitation, and soluble radioactivity was determined by scintillation counting. Experiments were performed in quadruplicate, and error bars substrate in the absence of enzyme (data not shown). 148 Vitronectin Induction of Matrix Metalloproteinase-2 experiments have demonstrated that adhesion of metastatic B16F1 and B16F10 cells; however, limited proteolysis of melanoma cells to lymph node sections is blocked by either vitronectin removed the stimulatory effect, regardless of anti-avb3 or RGD-containing peptides (3). In related experi- whether the RGD site was disrupted (plasmin) or remained ments, ligation of melanoma cell avb3 using an anti-integrin intact (endoproteinase V8). Furthermore, simple ligation of the antibody was shown to enhance cellular invasiveness in vitro, vitronectin receptor with the peptide RGDS also failed to in- and conditioned medium from cells treated with anti-avb3 duce MMP-2 activity at concentrations well in excess of that displayed increased MMP-2 activity (7). This is particularly required for inhibition of cell adhesion. However, antibody li- gation of melanoma cell avb3, which can induce receptor ag- interesting in light of previous data that indicate that MMP-2 expression by melanoma cells correlates with increased inva- gregation, stimulated MMP-2 production (7). Together, these siveness, and it provides a biochemical mechanism whereby data indicate that a multivalent ligand-receptor interaction, invasion may be enhanced (29 –31). rather than simple ligand occupancy, is required for induction These observations are supported by data from the present of MMP-2. In light of these results, it is interesting to consider recent biophysical data that demonstrate that under physio- study that demonstrate a direct dose-dependent increase in MMP-2 secretion in vitronectin-treated B16F1 and B16F10 logic conditions, vitronectin can exist in both monomeric and melanoma cells. As a functional consequence of increased multimeric forms (39). Vitronectin in extravascular sites (i.e. matrix and tissue-associated) is predominantly in the multim- MMP-2 levels, cellular invasiveness is also enhanced. The vitronectin-induced increase in MMP-2 secretion and invasive eric form, suggesting that multivalent ligand-receptor interac- activity was abolished by RGDS peptide, providing evidence tions may prevail in vivo (39, 40). Furthermore, the current data suggest a biologic control mechanism whereby the stimu- that vitronectin interaction with cellular integrins regulates invasive behavior. Concomitant with MMP-2 secretion, levels lus for MMP-2 induction, i.e. intact vitronectin, may be re- moved. It is interesting to speculate that vitronectin-adherent of TIMP-2 were also increased by vitronectin treatment. Pre- melanoma cells, which also catalyze tissue-type plasminogen vious studies have shown that MMP-2 is secreted as a proen- zyme in complex with TIMP-2 by melanoma cells and other cell activator-mediated plasmin generation (13), may initiate plas- min-dependent proteolysis of vitronectin, thereby disrupting types, and additional reports suggest that the presence of the multivalent signal necessary for MMP-2 induction. TIMP-2 in this proenzyme-inhibitor complex is required for cellular activation of the MMP-2 zymogen by membrane-type Acknowledgments—We thank Dr. C. N. 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Published: Jan 1, 1998

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