Osteopontin, a Novel Substrate for Matrix Metalloproteinase-3 (Stromelysin-1) and Matrix Metalloproteinase-7 (Matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity. Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity. osteopontin matrix metalloproteinase tumor necrosis factor α Chinese hamster ovary human OPN Osteopontin (OPN)1 is an arginine-glycine-aspartic acid (RGD)-containing glycoprotein that interacts with integrins and CD44 as major receptors. OPN has been shown to be multifunctional, with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function, and control of tumor cell phenotype (1Weber G.F. Cantor H. Cytokine Growth Factor Rev. 1996; 7: 241-248Crossref PubMed Scopus (118) Google Scholar, 2Denhardt D.T. Lopez C.A. Rollo E.E. Hwang S.M. An X.R. Walther S.E. Ann. N. Y. Acad. Sci. 1995; 760: 127-142Crossref PubMed Scopus (103) Google Scholar, 3Denhardt D.T. Chambers A.F. J. Cell. Biochem. 1994; 56: 48-51Crossref PubMed Scopus (92) Google Scholar, 4Uede T. Katagiri Y. Iizuka J. Murakami M. Microbiol. Immunol. 1997; 41: 641-648Crossref PubMed Scopus (90) Google Scholar). Targeting of the gene encoding OPN, spp1, has revealed that while OPN is not necessary for normal embryonic development, fertility, and health under pathogen-free conditions (5Liaw L. Birk D.E. Ballas C.B. Whitsitt J.S. Davidson J.M. Hogan B.L. J. Clin. Invest. 1998; 101: 1468-1478Crossref PubMed Google Scholar, 6Rittling S.R. Matsumoto H.N. McKee M.D. Nanci A. An X.R. Novick K.E. Kowalski A.J. Noda M. Denhardt D.T. J. Bone Miner. Res. 1998; 13: 1101-1111Crossref PubMed Scopus (371) Google Scholar), loss of the protein has significant consequences in several models of injury/disease as diverse as renal injury, viral and bacterial infection, bone remodeling, and tumor growth (7Ophascharoensuk V. Giachelli C.M. Gordon K. Hughes J. Pichler R. Brown P. Liaw L. Schmidt R. Shankland S.J. Alpers C.E. Couser W.G. Johnson R.J. Kidney Int. 1999; 56: 571-580Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 8Noiri E. Dickman K. Miller F. Romanov G. Romanov V.I. Shaw R. Chambers A.F. Rittling S.R. Denhardt D.T. Goligorsky M.S. Kidney Int. 1999; 56: 74-82Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Crawford H.C. Matrisian L.M. Liaw L. Cancer Res. 1998; 58: 5206-5215PubMed Google Scholar, 10Nau G.J. Liaw L. Chupp G.L. Berman J.S. Hogan B.L. Young R.A. Infect. Immun. 1999; 67: 4223-4230Crossref PubMed Google Scholar, 11Yoshitake H. Rittling S.R. Denhardt D.T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8156-8160Crossref PubMed Scopus (321) Google Scholar, 12Ashkar S. Weber G.F. Panoutsakopoulou V. Sanchirico M.E. Jansson M. Zawaideh S. Rittling S.R. Denhardt D.T. Glimcher M.J. Cantor H. Science. 2000; 287: 860-864Crossref PubMed Scopus (1002) Google Scholar). The fact that no other proteins seem to share a redundant activity with OPN under these conditions suggests that OPN has a unique functional role during tissue injury and stress. Interestingly, several members of the matrix metalloproteinase (MMP) family are also induced during injury/disease processes in patterns overlapping that of OPN (13McCawley L.J. Matrisian L.M. Mol. Med. Today. 2000; 6: 149-156Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). In particular, we have found that during squamous cell carcinoma progression, OPN and MMP-3 expression correspond both in a temporal and cell-specific fashion (9Crawford H.C. Matrisian L.M. Liaw L. Cancer Res. 1998; 58: 5206-5215PubMed Google Scholar, 14Wright J.H. McDonnell S. Portella G. Bowden G.T. Balmain A. Matrisian L.M. Mol. Carcinog. 1994; 10: 207-215Crossref PubMed Scopus (58) Google Scholar). We have also identified overlapping expression patterns of OPN and MMP-3 in the stroma during skin incisional wound healing (5Liaw L. Birk D.E. Ballas C.B. Whitsitt J.S. Davidson J.M. Hogan B.L. J. Clin. Invest. 1998; 101: 1468-1478Crossref PubMed Google Scholar) and OPN and MMP-7 during involution of the postpartum uterus (15Wilson C.L. Heppner K.J. Rudolph L.A. Matrisian L.M. Mol. Biol. Cell. 1995; 6: 851-869Crossref PubMed Scopus (134) Google Scholar). OPN is known to be a substrate for proteolytic cleavage by the proteases thrombin (16Yokosaki Y. Matsuura N. Sasaki T. Murakami I. Schneider H. Higashiyama S. Saitoh Y. Yamakido M. Taooka Y. Sheppard D. J. Biol. Chem. 1999; 274: 36328-36334Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 17Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (186) Google Scholar) and enterokinase (18Giachelli C.M. Liaw L. Murry C.E. Schwartz S.M. Almeida M. Ann. N. Y. Acad. Sci. 1995; 760: 109-126Crossref PubMed Scopus (174) Google Scholar). Thrombin cleavage of OPN (Arg168-Ser169 in humans, Arg153-Ser154 in rats) is of interest, since hydrolysis of this peptide bond reveals a binding site for the integrins α9β1 (19Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) and α4β1 (20Bayless K.J. Davis G.E. J. Biol. Chem. 2001; 276: 13483-13489Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), SVVYGLR, not present in the full-length molecule (16Yokosaki Y. Matsuura N. Sasaki T. Murakami I. Schneider H. Higashiyama S. Saitoh Y. Yamakido M. Taooka Y. Sheppard D. J. Biol. Chem. 1999; 274: 36328-36334Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). In addition, functional properties of thrombin-cleaved OPN differ from the intact protein (21O'Regan A.W. Chupp G.L. Lowry J.A. Goetschkes M. Mulligan N. Berman J.S. J. Immunol. 1999; 162: 1024-1031PubMed Google Scholar, 22Takahashi K. Takahashi F. Tanabe K.K. Takahashi H. Fukuchi Y. Biochem. Mol. Biol. Int. 1998; 46: 1081-1092PubMed Google Scholar, 23Senger D.R. Perruzzi C.A. Biochim. Biophys. Acta. 1996; 1314: 13-24Crossref PubMed Scopus (147) Google Scholar, 24Senger D.R. Ledbetter S.R. Claffey K.P. Papadopoulos-Sergiou A. Peruzzi C.A. Detmar M. Am. J. Pathol. 1996; 149: 293-305PubMed Google Scholar), demonstrating that proteolytic cleavage is one mechanism of regulating the bioactivity of OPN. In the present study, we have identified and characterized novel cleavage sites in OPN for two members of the MMP family, MMP-3 and MMP-7. Furthermore, we show that lower molecular weight forms of OPN corresponding to predicted MMP cleavage fragments are present in cell lines in vitro and in tissues in vivo. Biological assays demonstrate that the MMP-cleaved OPN has increased activity in promoting both cell adhesion and migration compared with full-length OPN. In addition, using inhibitory reagents, we have determined that the same receptors that interact with OPN also mediate interaction of MMP-cleaved OPN with tumor cells. These data suggest that active forms of OPN at sites of tissue injury may be regulated by the activity of proteases including MMPs and that the differences in activity of modified OPN may be explained by differences in binding affinity of integrins or distinct downstream signaling events. Recombinant rat OPN (LP432, CHO expression) and human OPN (LP462, Escherichia coliexpression) were generously provided by SmithKline Beecham. Active MMP-3 and MMP-7 were obtained from Chemicon, and the catalytic domain of MMP-3 (cat) was a kind gift of Dr. Hideaki Nagase (University of Kansas Medical Center). Thrombin was purchased from Sigma. The following antibodies were used as described. The anti-OPN antibodies were a goat polyclonal IgG OP199 (25Liaw L. Almeida M. Hart C.E. Schwartz S.M. Giachelli C.M. Circ. Res. 1994; 74: 214-224Crossref PubMed Scopus (380) Google Scholar) and rabbit polyclonals anti-human OPN, LP209, and LP210 (SmithKline Beecham). The anti-αvβ3 integrin clone LM609, anti-αvβ5 integrin clone P1F6, anti-α9β1 integrin clone Y9A2, anti-α4 integrin clone AV1, and anti-α5integrin clone NK1-SAM-1 were obtained from Chemicon. The peptides GRGDSP and GRGESP were obtained from Life Technologies, Inc. In some experiments, recombinant murine tumor necrosis factor α (TNFα, R&D Systems) was used at a concentration of 1 ng/ml. AsPC-1 and HeLa cells were obtained from ATCC and maintained in RPMI (Life Technologies) with 20% fetal bovine serum (AsPC-1) and minimum essential medium (Life Technologies, Inc.) with 10% fetal bovine serum. Primary mouse macrophages were obtained by peritoneal injection of 3% thioglycollate medium, with collection of exudate after 4 days. Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 50 µg/ml gentamycin. Primary chondrocytes were prepared from intervertebral discs as previously described (26Haro H. Crawford H.C. Fingleton B. MacDougall J.R. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 133-141Crossref PubMed Scopus (155) Google Scholar) and maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium with 10% fetal bovine serum, 25 µg/ml ascorbic acid, and 50 µg/ml gentamycin. Co-cultures of macrophages with chondrocytes were performed using cell culture inserts in six-well dishes with chondrocytes in the insert and macrophages in the wells as described (26Haro H. Crawford H.C. Fingleton B. MacDougall J.R. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 133-141Crossref PubMed Scopus (155) Google Scholar). Mo and Moαv subclones of M21 melanoma cells were kindly provided by Dr. Mark H. Ginsberg (Scripps Research Institute) and cultured as described. While Mo does not express αvintegrins, Moαv expresses high levels of αvintegrins at the cell surface (27Chen Y.P. O'Toole T.E. Leong L. Liu B.Q. Diaz-Gonzalez F. Ginsberg M.H. Blood. 1995; 86: 2606-2615Crossref PubMed Google Scholar). Rat and human OPN were cleaved by MMPs (enzyme/substrate ratio varied from 1:5 to 1:20 to maximize yield of cleavage fragments) and thrombin (0.25 units/µg of OPN) in equal volume of cleavage buffer (200 mm NaCl, 50 mm Tris-HCl, pH 7.6, 5 mm CaCl2) for 5–15 min at 37 °C. The amount of OPN was kept constant at 200 ng, and the enzyme amount varied according to the enzyme/substrate ratio used. For most biological assays, 10 ng of MMP was used with 200 ng of OPN for cleavage assays. The mixture of cleaved OPN fragments was separated on a 12.5% polyacrylamide gel under reducing conditions and then transferred to a 0.2-µm immunoblot polyvinylidene difluoride membrane (Bio-Rad). The membrane was washed with 50% methanol and stained with fresh Coomassie Brilliant Blue for less than 1 min and then destained with 50% methanol. Visible protein bands were excised by a razor-sharp blade and allowed to dry for about 1 h at room temperature and then shipped for sequencing. The N-terminal amino-terminal sequence was determined by modified automated Edman degradation (ProSeq Inc., Boxford, MA). For immunodetection, proteins were transferred to polyvinylidene difluoride membrane as above. For samples of chondrocyte/macrophage co-cultures, 22.5 µg of protein were present in each lane. For tumor cells and tissue lysates, 200 µg of protein were run per lane. The nonspecific binding was blocked with 10% nonfat dry milk in TBS-T buffer (10 mm Tris base, pH 8.0, 150 mm NaCl, and 0.1% Tween 20) at room temperature for 1 h. The membrane was incubated in a combination of three primary anti-OPN antibodies, OPN199 goat IgG (1:1000) and LP209 and LP210 rabbit IgG (1:2000), in 10% milk TBS-T for 1 h and then in horseradish peroxidase goat anti-rabbit and horseradish peroxidase rabbit anti-goat (1:2000) for another 1 h. The OPN bands were visualized using chemiluminescent reagent containing 250 mm 3-aminophthalydrazide and 90 mm p-coumaric acid. In situ hybridization on tissue sections was performed as previously described (9Crawford H.C. Matrisian L.M. Liaw L. Cancer Res. 1998; 58: 5206-5215PubMed Google Scholar) using sense and antisense riboprobes to the mouse OPN cDNA 2ar (28Craig A.M. Nemir M. Mukherjee B.B. Chambers A.F. Denhardt D.T. Biochem. Biophys. Res. Commun. 1988; 157: 166-173Crossref PubMed Scopus (113) Google Scholar). Adhesion and migration assays were performed as previously described (25Liaw L. Almeida M. Hart C.E. Schwartz S.M. Giachelli C.M. Circ. Res. 1994; 74: 214-224Crossref PubMed Scopus (380) Google Scholar). Briefly, for adhesion assays, test substrates were coated onto wells of Maxisorp 96-well plates and incubated overnight at 4 °C. After blocking with 10 mg/ml bovine serum albumin/Dulbecco's modified Eagle's medium for 1 h at 37 °C, wells were rinsed, and detached cells were plated for 1 h at 37 °C (AsPC-1 and HeLa cells plated at 30,000 cells/well and melanoma cell strains plated at 80,000 cells/well). Nonadherent cells were washed off, and attached cells were fixed with 4% paraformaldehyde, stained, and quantitated. Migration assays were performed using test substrates in the lower chamber of a modified Boyden chamber apparatus, separated from cells with a polycarbonate filter with 8-µm pores. After the migration period, cells on the upper surface of the filter (cells that did not migrate) were scraped off, and cells that had migrated through the pores to the lower surface of the membrane were fixed with 100% MeOH and stained with hematoxylin. Migrated cells were quantitated by cell counts of three random fields/well for six wells per test condition. For biological assays with protease cleaved OPN, cleavage of the substrate was verified by Western blot in all instances with an aliquot of the cleaved material before it was used for adhesion or migration assays. Inhibitory anti-integrin antibodies were used at a concentration of 25 µg/ml, and peptides were used at a concentration of 200 µg/ml. These reagents were incubated with cells for 15 min at 37 °C before cells were plated in adhesion assays. We have previously characterized a model of herniated disc resorption, allowing the study of the interaction of inflammatory cells with chondrocytes (26Haro H. Crawford H.C. Fingleton B. MacDougall J.R. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 133-141Crossref PubMed Scopus (155) Google Scholar). In this system, both MMP-3 and MMP-7 are activated (26Haro H. Crawford H.C. Fingleton B. MacDougall J.R. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 133-141Crossref PubMed Scopus (155) Google Scholar). MMP-3 is necessary for the generation of a macrophage chemoattractant (26Haro H. Crawford H.C. Fingleton B. MacDougall J.R. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 133-141Crossref PubMed Scopus (155) Google Scholar), while MMP-7 cleaves TNFα (29Haro H. Crawford H.C. Fingleton B. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 143-150Crossref PubMed Scopus (322) Google Scholar), and both events contribute to disc resorption. Because of the prevalence of OPN during inflammation, we were interested in possible interactions of these MMPs with OPN in this system. When we examined chondrocyte cultures in the presence or absence of macrophages, we found high levels of OPN in chondrocytes alone, but additional lower molecular weight forms detectable following co-culture with macrophages (Fig.1A). Since the co-culture system has been previously shown to induce the production of MMP-3 (26Haro H. Crawford H.C. Fingleton B. MacDougall J.R. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 133-141Crossref PubMed Scopus (155) Google Scholar), we tested whether MMP-3 might be responsible for the presence of the lower molecular weight forms of OPN. Using purified native rat OPN (25Liaw L. Almeida M. Hart C.E. Schwartz S.M. Giachelli C.M. Circ. Res. 1994; 74: 214-224Crossref PubMed Scopus (380) Google Scholar) and active MMP-3, cleavage assays were performed, and similar OPN bands at apparent molecular masses of 40 and 32 kDa were seen as a result of this cleavage (Fig. 1 B). In order to confirm the direct effect of MMP-3 in the co-cultures, two approaches were taken. Chondrocytes were cultured alone or in the presence of TNFα, a cytokine known to stimulate MMP-3 production (29Haro H. Crawford H.C. Fingleton B. Shinomiya K. Spengler D.M. Matrisian L.M. J. Clin. Invest. 2000; 105: 143-150Crossref PubMed Scopus (322) Google Scholar). In the absence of cytokines or macrophages, only full-length OPN was observed (Fig.1 C, lane 2), while a predominant 40-kDa band was additionally found in chondrocyte cultures treated with TNFα (lane 3). Furthermore, we utilized macrophages derived from wild type or MMP-3 null animals (30Mudgett J.S. Hutchinson N.I. Chartrain N.A. Forsyth A.J. McDonnell J. Singer I.I. Bayne E.K. Flanagan J. Kawka D. Shen C.F. Stevens K. Chen H. Trumbauer M. Visco D.M. Arthritis Rheum. 1998; 41: 110-121Crossref PubMed Scopus (182) Google Scholar) to confirm that MMP-3 was necessary for the generation of OPN fragments in the co-culture system. As shown in Fig. 1 C, chondrocytes co-cultured with macrophages from MMP-3 null (−/−) animals produced only the full-length OPN (lane 4; compare with chondrocytes cultured alone, (Fig. 1 A)), while co-cultures of chondrocytes and macrophages from wild type animals generated OPN cleavage fragments (lane 5). These data show that purified MMP-3 can cleave native OPN and that in the co-culture system, cleavage of endogenously produced OPN is dependent on the presence of endogenous MMP-3. To further characterize the proteolytic cleavage of OPN by MMPs, we studied recombinant OPN and active MMP-3 and MMP-7. We used recombinant human and rat OPN and found that both were substrates for MMP cleavage, although the cleavage pattern was slightly different (Fig.2). Similar to our results using native rat OPN (Fig. 1 A), recombinant rat OPN was cleaved by MMP-3 to generate two fragments at apparent molecular masses of 40 and 32 kDa (Fig. 2 A). However, the pattern of cleavage of recombinant human OPN (huOPN) differed (Fig. 2 B), and in addition to 40- and 32-kDa bands, MMP-3-cleaved huOPN additionally generated a 25-kDa band. Following MMP-7 cleavage of huOPN, 25-, 20-, and 15-kDa bands were generated. All MMP-cleaved OPN fragments were distinct from those generated by thrombin cleavage (Fig. 2 C), which generated 30- and 28-kDa bands in both rat and human recombinant OPN. Titration of enzyme/substrate ratios and cleavage times showed that with limiting amounts of enzyme and/or shorter cleavage times with MMP-7, huOPN also generated the 40- and 32-kDa bands, suggesting that these are intermediate forms in the reaction (Fig. 2 D). All cleavage products of both MMP-3 and MMP-7-cleaved rat and huOPN were analyzed by protein sequencing for determination of cleavage sites. The determined cleavage recognition sites are shown in Fig.3. The predominant cleavage site found for both MMP-3 and MMP-7 in rat and huOPN was a Gly-Leu bond (Gly166-Leu167 in huOPN, Gly151-Leu152 in rat OPN) just five amino acid residues C-terminal to the RGD sequence. Additionally, both MMP-3 and MMP-7 were found to cleave huOPN at the Asp210-Leu211 bond, but only MMP-3 displayed a minor cleavage site at Ala201-Tyr202 in huOPN. These cleavage sites corresponded with the apparent size of the fragments on SDS-polyacrylamide gel electrophoresis. A schematic showing cleavage patterns and OPN fragments generated is depicted in Fig. 4. At the present time, the only unidentified fragments that we expect should be generated are the small molecular weight fragments derived from MMP-3 cleavage of the 32-kDa band giving rise to the 25-kDa band and the MMP-7 cleavage of the 25-kDa band to generate the 20-kDa band (Fig. 4, question marks). These predicted fragments have not been detected even in high percentage gels, suggesting that they may be further degraded.Figure 3Identified MMP-3 and MMP-7 cleavage sites in rat and human OPN. Amino acid sequences of human and rat OPN are aligned, with cleavage sites for MMP-3, MMP-7, and thrombin indicated. Underlined sequences indicate α9β1 and α4β1recognition sites revealed following thrombin cleavage of OPN. The determined N-terminal sequence of each fragment is shown with the apparent molecular weight and corresponding fragments a–eas shown in Fig. 2.View Large Image Figure ViewerDownload (PPT)Figure 4Schematic OPN cleavage pathways for MMP-3 and MMP-7. OPN fragments with the apparent molecular weights listed as generated by MMP-3 or MMP-7 cleavage. Question marks indicate small fragments that have not been identified by SDS-polyacrylamide gel electrophoresis.View Large Image Figure ViewerDownload (PPT) To determine if other cell lines in addition to the chondrocyte and macrophage co-culture generated cleavage fragments of OPN, we screened a variety of tumor cell lines, which are known to have high expression of MMPs (31Meyer-Siegler K. Cytokine. 2000; 12: 914-921Crossref PubMed Scopus (36) Google Scholar, 32Attiga F.A. Fernandez P.M. Weeraratna A.T. Manyak M.J. Patierno S.R. Cancer Res. 2000; 60: 4629-4637PubMed Google Scholar, 33Ohnami S. Matsumoto N. Nakano M. Aoki K. Nagasaki K. Sugimura T. Terada M. Yoshida T. Cancer Res. 1999; 59: 5565-5571PubMed Google Scholar, 34Nagakawa O. Murakami K. Yamaura T. Fujiuchi Y. Murata J. Fuse H. Saiki I. Cancer Lett. 2000; 155: 173-179Crossref PubMed Scopus (65) Google Scholar). In addition, CHO cells that were either stably transfected with MMP-3 or vector alone were compared. Both conditioned medium and cell lysates were analyzed, and although OPN was detected in both compartments, there was consistently more protein detectable in the cell lysates. This was probably due to the fact that secreted OPN binds to cell surface proteoglycans and is found in the extracellular space (5Liaw L. Birk D.E. Ballas C.B. Whitsitt J.S. Davidson J.M. Hogan B.L. J. Clin. Invest. 1998; 101: 1468-1478Crossref PubMed Google Scholar), sometimes bound to other extracellular matrix proteins (35Mukherjee B.B. Nemir M. Beninati S. Cordella-Miele E. Singh K. Chackalaparampil I. Shanmugam V. DeVouge M.W. Mukherjee A.B. Ann. N. Y. Acad. Sci. 1995; 760: 201-212Crossref PubMed Scopus (63) Google Scholar). Thus, a large proportion of secreted OPN remained associated with the cell layer and extracellular matrix. The observation that a member of the MMP family, MMP-2, may associate with cell surface receptors (36Brooks P.C. Stromblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar) indicates a potential interaction of MMP with substrates at the cell surface or in the extracellular milieu. As shown in Fig. 5A, vector-transfected CHO cells did not produce detectable amounts of OPN protein by immunoblotting (lane 2). However, in CHO cells stably expressing MMP-3, OPN was detected in three forms, a faint species corresponding to full-length and two lower molecular weight forms running at 40 and 32 kDa (lanes 3and 4). In addition, in the tumor lines DU-145, a human prostate cancer cell line, and AsPC-1, a human pancreatic cancer line, multiple forms of OPN were detected, including bands corresponding to full-length OPN, as well as species with apparent molecular masses of 40, 32, 25, and 15 kDa (lanes 5–8). In vivo, the epithelial cells of the mouse postpartum uterus have been shown to be a region of high MMP-7 expression (15Wilson C.L. Heppner K.J. Rudolph L.A. Matrisian L.M. Mol. Biol. Cell. 1995; 6: 851-869Crossref PubMed Scopus (134) Google Scholar), in a pattern overlapping that of OPN expression (Fig. 5, B andC). MMP-7 and OPN expression overlap precisely in the epithelial cell layer of the remodeling uterus. Tissue extracts from 24-h mouse postpartum uterus (PPU) also showed strong lower molecular weight OPN bands in addition to the full-length protein (Fig.5 A). OPN is a well characterized adhesive protein and migratory stimulus. To test the regulation of bioactivity of OPN by MMP cleavage, adhesion and migration assays were performed using either recombinant full-length protein or OPN cleaved by MMP-3, MMP-7, or thrombin as a comparison. Full-length OPN was cleaved with MMPs or thrombin and used in adhesion assays in comparison with the same concentrations of OPN treated similarly but in the absence of enzyme. As a control, reactions were prepared with enzyme but in the absence of OPN (Fig.6A). HPLC-purified 40-kDa fragment was also used and gave similar results as the unpurified MMP-cleaved OPN. We found that the cleavage of OPN (either human or rat OPN gave similar results) with MMP-3, MMP-7, the catalytic domain of MMP-3 (cat), or thrombin significantly increased adhesion of tumor cells in comparison with full-length OPN alone. Interestingly, the sensitivity of the cells to protease-cleaved OPN was greatly enhanced compared with full-length OPN, as seen in dose comparison experiments (Fig. 6 A). Using 200 ng of OPN as a substrate, cleavage by MMP-3 and MMP-7 increased adhesion by ∼2-fold. However, with 50 ng of OPN as a substrate, MMP-3 and MMP-7 cleavage enhanced adhesion by 10- and 18-fold, respectively. Several cell lines were screened and found to display enhanced adhesion to MMP-cleaved OPN, including A5 and B9 murine squamous cell carcinoma lines, rat smooth muscle cells, human aortic endothelial cells, and NIH3T3 fibroblasts (p < 0.01 for all). Similarly, migration was tested using full-length OPN or MMP-3-cleaved OPN as a stimulus, and we found that macrophage migration toward MMP-3-cleaved OPN was significantly enhanced compared with full-length OPN (Fig. 6 B). Again, several cell types showed the same increased migratory response to MMP-3-cleaved OPN compared with full-length OPN, including A5 cells and B9 cells (p < 0.01), rat smooth muscle cells, human aortic endothelial cells, and NIH3T3 fibroblasts. These data show that the bioactivity of OPN toward cells can be regulated by proteolysis by MMPs. Although in many instances, the adhesive and migratory activity of MMP-cleaved OPN appeared similar to thrombin-cleaved OPN (Fig.6 A), we found situations in which the functional consequences of these proteolytic modifications were rather distinct. For example, HeLa carcinoma cell lines displayed a poor adhesion to full-length OPN and significantly higher, but not impressive, adhesion to MMP-cleaved OPN (Fig. 6 C). In contrast, high levels of adhesion were seen on a thrombin-cleaved OPN substrate. These findings imply that the activities of thrombin-cleaved versusMMP-cleaved OPN are distinct, and this may be related to receptor specificity on individual cell lines. One possibility explaining the enhanced bioactivity of MMP-cleaved OPN in comparison with full-length OPN is that different and additional receptors are activated. Indeed, this is the case with thrombin-cleaved OPN, where an α9β1 and α4β1 binding site is revealed after cleavage (19Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Since most OPN interaction with cells is integrin-mediated, we tested if known integrins were active in mediating cell responses to MMP-cleaved OPN. Using GRGDSP or GRGESP peptides, we found that interaction of the RGD sequence of either full-length OPN or MMP-cleaved OPN could account for virtually all of the adhesion of AsP