Osteopontin Stimulates Tumor Growth and Activation of Promatrix Metalloproteinase-2 through Nuclear Factor-κB-mediated Induction of Membrane Type 1 Matrix Metalloproteinase in Murine Melanoma Cells
2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês
10.1074/jbc.m103334200
ISSN1083-351X
AutoresSubha Philip, Anuradha Bulbule, Gopal C. Kundu,
Tópico(s)Peptidase Inhibition and Analysis
ResumoMatrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and play critical roles in tissue repair, tumor invasion, and metastasis. MMPs are regulated by different cytokines, ECM proteins, and other factors. However, the molecular mechanisms by which osteopontin (OPN), an ECM protein, regulates ECM invasion and tumor growth and modulates MMP activation in B16F10 cells are not well defined. We have purified OPN from human milk and shown that OPN induces pro-MMP-2 production and activation in these cells. Moreover, our data revealed that OPN-induced membrane type 1 (MT1) MMP expression correlates with translocation of p65 (nuclear factor-κB (NF-κB)) into the nucleus. However, when the super-repressor form of IκBα (inhibitor of NF-κB) was transfected into cells followed by treatment with OPN, no induction of MT1-MMP expression was observed, indicating that OPN activates pro-MMP-2 via an NF-κB-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with αvβ3integrin, but these effects were reduced drastically when the MMP-2-specific antisense S-oligonucleotide was used to suppress MMP-2 expression. Interestingly, when the OPN-treated cells were injected into nude mice, the mice developed larger tumors, and the MMP-2 levels in the tumors were significantly higher than in controls. The proliferation data indicate that OPN increases the growth rate in these cells. Both tumor size and MMP-2 expression were reduced dramatically when anti-MMP-2 antibody or antisenseS-oligonucleotide-transfected cells were injected into the nude mice. To our knowledge, this is the first report that MMP-2 plays a direct role in OPN-induced cell migration, invasion, and tumor growth and that demonstrates that OPN-stimulated MMP-2 activation occurs through NF-κB-mediated induction of MT1-MMP. Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and play critical roles in tissue repair, tumor invasion, and metastasis. MMPs are regulated by different cytokines, ECM proteins, and other factors. However, the molecular mechanisms by which osteopontin (OPN), an ECM protein, regulates ECM invasion and tumor growth and modulates MMP activation in B16F10 cells are not well defined. We have purified OPN from human milk and shown that OPN induces pro-MMP-2 production and activation in these cells. Moreover, our data revealed that OPN-induced membrane type 1 (MT1) MMP expression correlates with translocation of p65 (nuclear factor-κB (NF-κB)) into the nucleus. However, when the super-repressor form of IκBα (inhibitor of NF-κB) was transfected into cells followed by treatment with OPN, no induction of MT1-MMP expression was observed, indicating that OPN activates pro-MMP-2 via an NF-κB-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with αvβ3integrin, but these effects were reduced drastically when the MMP-2-specific antisense S-oligonucleotide was used to suppress MMP-2 expression. Interestingly, when the OPN-treated cells were injected into nude mice, the mice developed larger tumors, and the MMP-2 levels in the tumors were significantly higher than in controls. The proliferation data indicate that OPN increases the growth rate in these cells. Both tumor size and MMP-2 expression were reduced dramatically when anti-MMP-2 antibody or antisenseS-oligonucleotide-transfected cells were injected into the nude mice. To our knowledge, this is the first report that MMP-2 plays a direct role in OPN-induced cell migration, invasion, and tumor growth and that demonstrates that OPN-stimulated MMP-2 activation occurs through NF-κB-mediated induction of MT1-MMP. matrix metalloproteinases tissue inhibitor of matrix metalloproteinase extracellular matrix osteopontin membrane type 1 nuclear factor-κB MMP-2-specific antisenseS-oligonucleotide MMP-2-specific senseS-oligonucleotide fast protein liquid chromatography polyacrylamide gel electrophoresis phosphate-buffered saline penicillamine Cell migration and extracellular matrix invasion are some of the major steps in embryonic development (1Strickland S. Riech E. Sherman M.I. Cell. 1976; 9: 231-240Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 2Sappino A.P. Huarte D. Vassalli J.D. J. Cell Biol. 1989; 109: 2471-2479Crossref PubMed Scopus (232) Google Scholar) and wound healing and cancer cell metastasis (3Liotta L.A. Steeg P.S. Steller-Stevenson W.G. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 4Testa J.E. Quigley J.P. Cancer Metastasis Rev. 1990; 9: 353-367Crossref PubMed Scopus (152) Google Scholar). However, the exact molecular mechanisms that regulate these processes are not well understood. In the past, several investigators have shown that matrix metalloproteinases (MMPs)1 and the tissue inhibitor of matrix metalloproteinase (TIMP) play a major role in the regulation of cancer cell migration, extracellular matrix (ECM) invasion, and metastasis by degrading the ECM proteins (3Liotta L.A. Steeg P.S. Steller-Stevenson W.G. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 4Testa J.E. Quigley J.P. Cancer Metastasis Rev. 1990; 9: 353-367Crossref PubMed Scopus (152) Google Scholar, 5Murphy G. Gavrilovic J. Curr. Opin. Cell Biol. 1999; 11: 614-621Crossref PubMed Scopus (345) Google Scholar). Current investigations have focused on the understanding of molecular mechanism(s) by which osteopontin (OPN), an ECM protein, regulates MMP expression both in vitro and in vivo and controls invasiveness and tumor growth in B16F10 cells. OPN is a noncollagenous, sialic acid-rich, and glycosylated phosphoprotein (6Butler W.T. Connect. Tissue Res. 1989; 23: 123-136Crossref PubMed Scopus (486) Google Scholar, 7Denhardt D. Guo X. FASEB J. 1993; 7: 1475-1482Crossref PubMed Scopus (1005) Google Scholar). It has an N-terminal signal sequence, a highly acidic region consisting of nine consecutive aspartic acid residues, and a GRGDS cell adhesion sequence predicted to be flanked by the β-sheet structure (8Prince C.W. Connect. Tissue Res. 1989; 21: 15-20Crossref PubMed Scopus (57) Google Scholar). This protein has a functional thrombin cleavage site and is a substrate for tissue transglutaminase (7Denhardt D. Guo X. FASEB J. 1993; 7: 1475-1482Crossref PubMed Scopus (1005) Google Scholar). OPN binds with type I collagen (9Chen Y. Bal B.S. Gorski J.P. J. Biol. Chem. 1992; 267: 24871-24878Abstract Full Text PDF PubMed Google Scholar), fibronectin (10Singh K. Devouge M.W. Mukherjee B.B. J. Biol. Chem. 1990; 265: 18696-18701Abstract Full Text PDF PubMed Google Scholar), and osteocalcin (11Ritter N.M. Farach-Carson M.C. Butler W.T. J. Bone Miner. Res. 1992; 7: 877-885Crossref PubMed Scopus (101) Google Scholar). Several highly metastatic transformed cells synthesize higher levels of OPN compared with non-tumorigenic cells (12Craig A.M. Bowden G.T. Chambers A.F. Spearman M.A. Greenberg A.H. Wright J.A. Denhardt D.T. Int. J. Cancer. 1990; 46: 133-137Crossref PubMed Scopus (113) Google Scholar). It has been shown that OPN also interacts with CD44 receptor globulin (13Weber G.F. Akshar S. Glimcher M.J. Cantor H. Science. 1996; 271: 509-512Crossref PubMed Scopus (809) Google Scholar). OPN causes cell adhesion and migration, ECM invasion, and cell proliferation by interacting with its receptor αvβ3 integrin in various cell types (14Panda D. Kundu G.C. Lee B.I. Peri A. Fohl D. Chackalaparampil I. Mukherjee B.B. Li X.D. Mukherjee D.C. Seides S. Rosenberg J. Stark K. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9308-9313Crossref PubMed Scopus (153) Google Scholar). Integrins are noncovalently associated, heterodimeric, cell-surface glycoproteins with α- and β-subunits. Integrins are a superfamily of transmembrane glycoproteins found predominantly on the surface of leukocytes that mediate cell-cell and cell-substratum interactions. Until today, ∼12 α-subunits, 8 β-subunits, and 20 αβ-heterodimers were documented in the literature (15Giancotti F.G. Rhuoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3794) Google Scholar). MMPs are a family of enzymes that are classified under different subgroups (16Nagase H. Woessner J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar). MMP-2 (also called type IV collagenase or gelatinase A) degrades several ECM proteins such as fibronectin, laminin, type I collagen, and proteoglycans. It plays critical roles in embryogenesis, tissue remodeling, inflammation, periodontitis, and metastasis (16Nagase H. Woessner J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar). Several reports have indicated that the increased levels of MMP-2 correlate with the invasive properties of certain tumor cell types (16Nagase H. Woessner J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar,17Seftor R.E.B. Seftor E.A. Gehlsen K.R. Stetler-Stevenson W.G. Brown P.D. Ruoslahti E. Hendrix M.J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1557-1561Crossref PubMed Scopus (433) Google Scholar). TIMP-2 is the specific inhibitor of MMP-2. TIMP-2 is a non-glycosylated protein (21 kDa) that forms a complex with both the inactive and active forms of MMP-2 (18Miyazaki K. Funahashi K. Numata Y. Koshikawa N. Akaogi K. Kikkawa Y. Yasumitsu H. Umeda M. J. Biol. Chem. 1993; 268: 14387-14393Abstract Full Text PDF PubMed Google Scholar). Previous studies have demonstrated that the ligation of αvβ3 integrin in melanoma cells by anti-αvβ3 integrin antibodies enhances the expression of MMP-2, resulting in increased levels of cellular invasiveness (17Seftor R.E.B. Seftor E.A. Gehlsen K.R. Stetler-Stevenson W.G. Brown P.D. Ruoslahti E. Hendrix M.J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1557-1561Crossref PubMed Scopus (433) Google Scholar). Furthermore, treatment of melanoma cells with vitronectin induces the expression of MMP-2 and TIMP-2 as well as enhances cellular invasiveness in a dose-dependent manner (19Bafetti L.M. Young T.N. Itoh Y. Stack M.S. J. Biol. Chem. 1998; 273: 143-149Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Takahashi et al. (20Takahashi K. Eto H. Tanabe K.K. Int. J. Cancer. 1999; 80: 387-395Crossref PubMed Scopus (85) Google Scholar) showed that treatment of human melanoma cells with monoclonal anti-CD44 antibody induced the expression of MMP-2 and enhances cell migration and ECM invasion. However, treatment of the same melanoma cells with recombinant OPN-glutathione S-transferase fusion protein failed to induce the expression of MMP-2. It was speculated that MMP-2 expression did not occur probably because of the glutathioneS-transferase fusion with osteopontin (20Takahashi K. Eto H. Tanabe K.K. Int. J. Cancer. 1999; 80: 387-395Crossref PubMed Scopus (85) Google Scholar). Recently, Maquoiet al. (21Maquoi E. Frankenne F. Noel A. Krell H.-W. Grams F. Foidart J.-M. Exp. Cell Res. 2000; 261: 348-359Crossref PubMed Scopus (60) Google Scholar) have shown that type IV collagen induces the activation of MMP-2 in human fibrosarcoma cells. Here we report that treatment of B16F10 cells with purified human OPN (but not with other ECM proteins such as fibronectin, type I collagen, and laminin) induced the expression of pro-MMP-2 and active MMP-2. We provide evidence that OPN induced membrane type 1 (MT1) MMP expression by stimulating the nuclear factor-κB (NF-κB) pathway. OPN also enhanced cell migration and ECM invasion by interacting with the αvβ3 integrin receptor in these cells. Transient transfection of B16F10 cells with the MMP-2-specific antisense S-oligonucleotide (ASMMP-2), but not with the sense S-oligonucleotide (SMMP-2), caused pronounced inhibition of MMP-2 protein expression and drastic suppression of OPN-induced cell migration and ECM invasion. Interestingly, OPN also induced tumor growth, and expression of MMP-2 in tumors of OPN-injected nude mice was significantly higher than in controls. Moreover, when anti-MMP-2 antibody was injected into OPN-induced tumors, the size of the tumors and the levels of active MMP-2 were reduced dramatically compared with controls. Similarly, when ASMMP-2-transfected cells were injected into the mice, the size of the tumors and the levels of MMP-2 were also suppressed compared with SMMP-2- or LipofectAMINE Plus-injected cells. Taken together, these data indicate that OPN-induces pro-MMP-2 expression and activation and enhances cell migration, ECM invasion, and tumor growth. These data further demonstrate that MMP-2 is mechanistically involved in the regulation of these processes. Fibronectin, type I collagen, laminin, GRGDSP, GPenGRGDSPCA, GRGESP, and LipofectAMINE Plus reagent were purchased from Life Technologies, Inc. Mouse monoclonal anti-MMP-2 (Ab-3) and anti-MT1-MMP antibodies were obtained from Oncogene Research. Rabbit anti-human αvβ3 integrin antibody was from Chemicon International. Goat anti-human αv integrin, mouse monoclonal anti-β3integrin, and rabbit polyclonal anti-NF-κB p65 antibodies were obtained from Santa Cruz Biotechnology. The IgG-purified anti-OPN antibody was purchased from R&D Systems. The anti-OPN antibody was also raised against purified intact human OPN in rabbits and characterized in our laboratory. Na125I (carrier-free; 3.7 GBq/ml) was purchased from Board of Radiation and Isotope Technology (Mumbai, India). Boyden-type cell migration chambers were obtained from Corning Corp. BioCoat MatrigelTM invasion chambers were from Collaborative Biomedical. The nude mice (NMRI, nu/nu) were obtained from the National Institute of Virology (Pune, India). All other chemicals were analytical grade. The murine melanoma cells (B16F10) were obtained from American Type Culture Collection (Manassas, VA). These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Human milk was collected from a local hospital. OPN was purified by DEAE-Sephadex chromatography, followed by FPLC. Briefly, the milk sample was centrifuged, and the cleared sample was loaded onto the DEAE-Sephadex column. The column was washed with 10 mm sodium phosphate buffer (pH 7.0) containing 100 mm NaCl and eluted with the same buffer containing 500 mm NaCl. The fractions containing OPN were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) according to Laemmli (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar), followed by Western blot analysis. Briefly, the OPN sample was resolved by SDS-PAGE and electrotransferred from the gel to a nitrocellulose membrane. The membrane was incubated with established IgG-purified anti-OPN antibody (1:5000 dilution). It was washed, incubated further with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000 dilution), and detected using an ECL detection system (Amersham Pharmacia Biotech). Partially purified OPN was purified further on an FPLC Resource-Q column with a linear gradient of 0–1 m NaCl in 20 mm Tris-HCl (pH 7.6) over a period of 40 min and rechromatographed under the same conditions on the Resource-Q column. The final purity of OPN was checked by SDS gel and Western blot analyses using both established and in-house anti-OPN antibodies as described above. The concentration of OPN was measured by the Bradford method (Bio-Rad protein assay) according to the manufacturer's instructions. To check the effect of OPN on MMP-2 expression and activation, the B16F10 cells were treated with varying concentrations of purified OPN (0–10.0 μm) in serum-free medium and incubated at 37 °C for 24 h. The conditioned media were collected by centrifugation, concentrated, and dialyzed. The dialyzed samples containing an equal amount of total proteins were mixed with sample buffer in the absence of reducing agent, incubated at room temperature for 30 min, and loaded onto zymographic SDS gel containing gelatin (0.5 mg/ml) as described (23Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-528Crossref PubMed Scopus (243) Google Scholar). The gels were washed and incubated in incubation buffer (50 mm Tris-HCl (pH 7.5) containing 100 mm CaCl2, 1 μmZnCl2, 1% (v/v) Triton X-100, and 0.02% (w/v) NaN3) for 16 h. The gels were stained with Coomassie Blue and destained. The zones of gelatinolytic activity were shown by negative staining. Purified MMP-2 was used as a control. In separate experiments, the cells were treated with other ECM proteins (5.0 μm each fibronectin, type I collagen, or laminin) or peptides (10.0 μm each GRGDSP, GPenGRGDSPCA, or GRGESP) and incubated under the same conditions described above. The conditioned media were collected and used for zymography. To analyze the levels of MMP-2 and TIMP-2 mRNAs in untreated and OPN-treated cells, the cells were first treated with OPN (10 μm), and total RNA was extracted using the RNAzol method (Tel-Test) according to the manufacturer's instructions. RNA concentration was measured spectrophotometrically at absorbances of 260 and 280 nm. An equal amount of total RNA (20 μg) was denatured in formaldehyde and resolved by electrophoresis on formaldehyde-agarose gels. The RNA samples were transferred from the gels to nylon membranes and cross-linked by UV irradiation. Hybridization was performed using an α-32P-labeled mouse MMP-2 or TIMP-2 probe. The 708-base pair MMP-2 cDNA probe was obtained by polymerase chain reaction amplification, followed by hybridization with an MMP-2-specific probe. The 508-base pair TIMP-2 probe was obtained by digesting plasmid pBluescript SK containing partial-length TIMP-2 cDNA (nucleotides 1173–1680) (a generous gift from Prof. M. Seiki, University of Tokyo, Tokyo, Japan) with HindIII. The DNA was purified by agarose gel electrophoresis. The probe was labeled with [α-32P]dCTP using a random primer labeling kit (Bangalore Genei, Bangalore, India) according to the manufacturer's instructions. The blots were exposed to Eastman Kodak X-Omat AR x-ray film and autoradiographed. The nuclear extracts were prepared using a modification (24Lee K.A. Bindereif A. Green M.R. Gene Anal. Tech. 1988; 5: 22-31Crossref PubMed Scopus (394) Google Scholar) of the method of Dignam (40Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9149) Google Scholar). Briefly, cells were incubated in the absence or presence of OPN (5 μm) in serum-free medium at 37 °C for 24 h. Cells were scraped, washed with PBS, resuspended in hypotonic buffer (10 mm Hepes (pH 7.9), 10 mm MgCl2, 10 mm KCl, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mmdithiothreitol), and allowed to swell on ice for 10 min. Cells were homogenized in a Dounce homogenizer. The nuclei were separated by spinning at 3300 × g for 5 min at 4 °C and resuspended in low salt buffer (20 mm Hepes (pH 7.9), 25% glycerol, 1.5 mm MgCl2, 1.2 mm KCl, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mm dithiothreitol), followed by the addition of an equal volume of high salt buffer. The lysed nuclei were centrifuged at 12,000 × g for 5 min at 4 °C, and the cleared supernatant was collected. The post-nuclear supernatant was used for cytoplasmic extraction. To this supernatant was added 0.1 volume of cytoplasm extraction buffer (0.3 mm Hepes (pH 7.9) containing 30 mm MgCl2 and 1.4m KCl). The sample was centrifuged, and the cleared supernatant was collected. The protein concentrations in the supernatants of both nuclear and cytoplasmic extracts were measured by the Bio-Rad protein assay. The level of NF-κB p65 in both nuclear and cytoplasmic extracts was detected by Western blot analysis using rabbit polyclonal anti-NF-κB p65 antibody as described above. In other experiments, the super-repressor form of IκBα fused downstream to a FLAG epitope in an expression vector (pCMV4) was transiently transfected into cells using LipofectAMINE Plus reagent according to the manufacturer's instructions. Briefly, the vector containing IκBα was mixed with plus reagent in Opti-MEM I and incubated at room temperature for 15 min. LipofectAMINE was mixed with DNA plus reagent and incubated further at room temperature for 15 min. The LipofectAMINE Plus-DNA complex was added to cells and mixed by gentle agitation. After 48 h, the cells were treated in the absence or presence of OPN (5 μm) and incubated further at 37 °C for 24 h. Cells were extracted with radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mm iodoacetamide, and 2 mm phenylmethylsulfonyl fluoride) at 4 °C for 2 h. The supernatant was collected by centrifugation and used for detection of MT1-MMP by Western blotting using mouse monoclonal anti-MT1-MMP antibody as described above. The migration assay was conducted using a Transwell cell culture chamber according to the standard procedure described (25Yue T.L. McKenna P.J. Ohlstein E.H. Farach-Carson M.C. Butler W.T. Johanson K. McDevitt P. Feuerstein G.Z. Stadel J.M. Exp. Cell Res. 1994; 214: 459-464Crossref PubMed Scopus (128) Google Scholar). Briefly, the confluent monolayer of B16F10 cells was harvested with trypsin/EDTA and centrifuged at 800 ×g for 10 min. The cell suspension (5 × 105cells/well) was added to the upper chamber of a pre-hydrated polycarbonate membrane filter. Purified intact OPN (0–10 μm) was added to the upper chamber. The lower chamber was filled with fibroblast-conditioned medium, which acted as a chemoattractant. To ascertain whether OPN-stimulated migration occurred via αvβ3 integrin, the cells were pretreated with anti-human αvβ3 integrin antibody (40 μg/ml) for 20 min, and then OPN was added (5.0 μm). Similarly, the cells were also pretreated with monoclonal anti-MMP-2 antibody (50.0 μg/ml), and then OPN was added. In other experiments, the cells were individually treated with GRGDSP, GRGESP, or GPenGRGDSPCA (5.0 μm each) for 20 min and then incubated with OPN (5.0 μm). After treatment, the cells were incubated in a humidified incubator in 5% CO2 and 95% air at 37 °C for 24 h. The non-migrating cells on the upper side of the filter were scraped and washed. The migrating cells on the reverse side of the filter were stained with Giemsa. The migrating cells on the filter were counted and a photomicrograph was taken under an Olympus inverted microscope. In separate experiments, SMMP-2- or ASMMP-2-transfected cells (5 × 105cells/well) were added to the upper chamber, and a migration assay was performed under the same conditions as described above. The experiments were repeated in triplicate. Preimmune IgG served as a nonspecific control. The ECM invasion assay was performed using commercially available 24-well plates that consist of upper and lower chambers as described (26Melchiorri A. Carlone S. Allanvena G. Aresu O. Parodi S. Aaronson S. Albini A. Anticancer Res. 1990; 10: 37-44PubMed Google Scholar, 27Kundu G.C. Mantile G. Miele L. Cordella-Miele E. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2915-2919Crossref PubMed Scopus (61) Google Scholar). The two chambers were divided by a porous filter, the upper surface of which was precoated with a layer of artificial basement membrane, MatrigelTM (Collaborative Biomedical). The cell suspension (5 × 105 cells/well) was added to the upper chamber. The lower chamber was filled with fibroblast-conditioned medium. which acted as a chemoattractant. The cells were treated with varying concentrations of purified intact OPN (0–10.0 μm) and then added to the upper chamber. In some experiments, the cells were pretreated with anti-human αvβ3 integrin antibody (40.0 μg/ml), monoclonal anti-MMP-2 antibody (50.0 μg/ml), GRGDSP (5.0 μm), GRGESP (5.0 μm), or GPenGRGDSPCA (5.0 μm) for 20 min and then incubated with OPN (5.0 μm) at 37 °C for 24 h as described above. The non-migrating cells and Matrigel from the upper side of the filter were scraped and removed using a moist cotton swab. The invaded cells in the lower side of the filter were stained with Giemsa and washed with PBS (pH 7.6). The invaded cells were then counted, and photomicrographs were taken under the Olympus inverted microscope. In other experiments, SMMP-2- or ASMMP-2-transfected cells (5 × 105cells/well) were added to the upper chamber, and the invasion assay was done under the same conditions as described above. The experiments were repeated in triplicate. Preimmune IgG was used as a nonspecific control. The proliferation assay was performed as described previously (14Panda D. Kundu G.C. Lee B.I. Peri A. Fohl D. Chackalaparampil I. Mukherjee B.B. Li X.D. Mukherjee D.C. Seides S. Rosenberg J. Stark K. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9308-9313Crossref PubMed Scopus (153) Google Scholar). Briefly, serum-starved B16F10 cells were incubated in the absence or presence of increasing concentrations of OPN (0–10 μm) at 37 °C for 24 h. After 4 h, [3H]thymidine (1 μCi/ml) was added, and the cells were maintained in culture for another 20 h. After removing the supernatants, the cells were washed with basal medium and lysed in 50% trichloroacetic acid. The acid-precipitable cell-bound radioactivity was measured with a scintillation counter (Packard Instrument Co.). To detect the level of αvβ3 integrin in B16F10 cells, the cells were surface-labeled with Na125I and IODO-BEADS as described (28O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar). The cells were lysed in lysis buffer composed of 1% Triton X-100 solution containing 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 2 mm EDTA and immunoprecipitated individually with anti-human αv or monoclonal anti-β3 integrin antibody (Roche Molecular Biochemicals) according to the manufacturer's instructions. The samples were boiled in SDS sample buffer, electrophoresed, and autoradiographed. Purified human OPN (20 μg) was radioiodinated using Na125I (carrier-free; 2 mCi) and chloramine T as described (29Hunter W.M. Greenwood F.C. Nature. 1962; 194: 495-496Crossref PubMed Scopus (5859) Google Scholar). 125I-OPN was purified by Sephadex G-50 column chromatography, and radioactivity was measured in a γ-counter (Beckman Instruments) with a counting efficiency of ∼85%. The specific activity of carrier-free purified monoiodinated OPN was 78 μCi/μg. For binding assays, subconfluent cultures of B16F10 cells were incubated with 125I-OPN (3.0 × 105 cpm/well) in the absence or presence of increasing concentrations of unlabeled purified OPN in 1.0 ml of Hanks' balanced salt solution (pH 7.6) containing 0.1% bovine serum albumin. After incubation at 37 °C for 2 h, the reactions were stopped by rapid removal of medium containing unbound radiolabeled OPN. The cells were solubilized with 1 n NaOH and neutralized with 1n HCl, and radioactivity was measured in the γ-counter. Specific binding was calculated by subtracting nonspecific binding from total binding. The Kd value was determined by Scatchard analysis using the LIGAND computer program (30Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7771) Google Scholar). Murine ASMMP-2 (5′-CCA CTC GTG CCT CCA TCG TT-3′) and SMMP-2 (5′-AAC GAT GGA GGC ACG AGT-3′) oligonucleotides with phosphorothioate linkages were synthesized (Gemini Biotech). These oligonucleotides were purified by column chromatography, and purity was checked by polyacrylamide gel electrophoresis. The B16F10 cells were pretreated with OPN (10 μm) at 37 °C for 6 h and then transfected transiently with the sense or antisense S-oligonucleotide using LipofectAMINE Plus according to the manufacturer's instructions. Briefly, the sense or antisense S-oligonucleotide was mixed with plus reagent, and then oligonucleotide reagent plus was incubated with LipofectAMINE. The LipofectAMINE Plus-oligonucleotide complex was added to the cells and incubated further at 37 °C for 8 h. The control cells received LipofectAMINE Plus alone. The cell viability was detected by a trypan blue dye exclusion test. After incubation, the oligonucleotide-containing medium was removed, and the cells were refed with fresh medium and cultured for an additional 12 h. The serum-free conditioned medium was used for the detection of MMP-2 by zymography, and the cells were used for migration, ECM invasion, and tumorigenicity experiments as described above. To check the dose-dependent response, a separate transfection experiment was performed with different doses (0–10 μg) of ASMMP-2. The tumorigenicity experiments were performed as described (31Sun Y. Kim H. Parker M. Stetler-Stevenson W.G. Colburn N.H. Anticancer Res. 1996; 16: 1-17PubMed Google Scholar). The cells were grown in monolayer and treated in the absence or presence of purified human OPN (10 μm) in serum-free medium. The cells were incubated at 37 °C for 24 h. After that, the cells (5 × 106 cells/0.2 ml) were detached, centrifuged, washed, and injected subcutaneously into the flanks of male athymic NMRI (nu/nu) mice (6–8 weeks old). Four mice were used in each set of experiments. The mice were kept under specific pathogen-free conditions. OPN (10 μm) was again injected into the tumor sites twice a week for up to 4 weeks. In other experiments, the cells were treated with OPN (10 μm) and injected into mice. A mixture (0.2 ml) of anti-MMP-2 antibody (50 μg/ml) and OPN (10.0 μm) was injected into the same tumor sites twice a week for up to 4 weeks. In separate experiments, the LipofectAMINE Plus-oligonucleotide (ASMMP-2 or SMMP-2) complex was injected into the tumors of the nude mice. After 4 weeks, the mice were killed, and the tumor weights were measured. The tumor tissues were homogenized; l
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