Extracellular Matrix Metalloproteinase 2 Levels Are Regulated by the Low Density Lipoprotein-related Scavenger Receptor and Thrombospondin 2
We have recently shown that the adhesive defect observed in dermal fibroblasts derived from thrombospondin 2 (TSP2)-null mice results from an increase in matrix metalloproteinase 2 (MMP2) levels (Yang, Z., Kyriakides, T. R., and Bornstein, P. (2000) Mol. Biol. Cell 11, 3353–3364). Adhesion was restored by replacement of TSP2 and by inhibitors of MMP2 activity. In pursuing the observation that TSP2 and MMP2 interact, we now demonstrate that this interaction is required for optimal clearance of extracellular MMP2 by fibroblasts. Since TSP2 is known to be endocytosed by the scavenger receptor, low density lipoprotein receptor-related protein (LRP), we determined whether interference with LRP function affected fibroblast adhesion and/or extracellular MMP2 levels. Addition of heparin, which competes for the binding of TSP2 to LRP coreceptor proteoglycans, inhibited adhesion of control but not TSP2-null cells, and a blocking antibody to LRP as well as the LRP inhibitor, receptor-associated protein, also inhibited adhesion and increased MMP2 levels only in control fibroblasts. TSP2 did not inhibit active MMP2 directly and did not inhibit the activation of pro-MMP2. Finally, the internalization of 125I-MMP2 was reduced in TSP2-null compared with control fibroblasts. We propose that clearance of MMP2-TSP2 complexes by LRP is an important mechanism for the regulation of extracellular MMP2 levels in fibroblasts, and perhaps in other cells. Thus, some features of the phenotype of TSP2-null mice, such as abnormal collagen fibrillogenesis, accelerated wound healing, and increased angiogenesis, could result in part from increased MMP2 activity. We have recently shown that the adhesive defect observed in dermal fibroblasts derived from thrombospondin 2 (TSP2)-null mice results from an increase in matrix metalloproteinase 2 (MMP2) levels (Yang, Z., Kyriakides, T. R., and Bornstein, P. (2000) Mol. Biol. Cell 11, 3353–3364). Adhesion was restored by replacement of TSP2 and by inhibitors of MMP2 activity. In pursuing the observation that TSP2 and MMP2 interact, we now demonstrate that this interaction is required for optimal clearance of extracellular MMP2 by fibroblasts. Since TSP2 is known to be endocytosed by the scavenger receptor, low density lipoprotein receptor-related protein (LRP), we determined whether interference with LRP function affected fibroblast adhesion and/or extracellular MMP2 levels. Addition of heparin, which competes for the binding of TSP2 to LRP coreceptor proteoglycans, inhibited adhesion of control but not TSP2-null cells, and a blocking antibody to LRP as well as the LRP inhibitor, receptor-associated protein, also inhibited adhesion and increased MMP2 levels only in control fibroblasts. TSP2 did not inhibit active MMP2 directly and did not inhibit the activation of pro-MMP2. Finally, the internalization of 125I-MMP2 was reduced in TSP2-null compared with control fibroblasts. We propose that clearance of MMP2-TSP2 complexes by LRP is an important mechanism for the regulation of extracellular MMP2 levels in fibroblasts, and perhaps in other cells. Thus, some features of the phenotype of TSP2-null mice, such as abnormal collagen fibrillogenesis, accelerated wound healing, and increased angiogenesis, could result in part from increased MMP2 activity. thrombospondin low density lipoprotein-related receptor protein matrix metalloproteinase 2 receptor-associated protein 4-aminophenylmercuric acetate polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium Thrombospondins (TSP)1 1 and 2 are large extracellular macromolecules whose diverse functions reflect their ability to bind to multiple cell-surface receptors, cytokines, growth factors, and proteases, and to structural components of the matrix (1Lawler J. Curr. Opin. Cell Biol. 2000; 12: 634-640Crossref PubMed Scopus (372) Google Scholar, 2Bornstein P. Armstrong L.C. Hankenson K.D. Kyriakides T.R. Yang Z. Matrix Biol. 2000; 19: 557-568Crossref PubMed Scopus (135) Google Scholar). TSP2-null mice display a complex phenotype that is characterized by changes in connective tissues, particularly in response to injury, an increase in vascular density and endosteal bone growth, and a bleeding defect (3Kyriakides T.R. Zhu Y.-H. Smith L.T. Bain S.D. Yang Z. Lin M.T. Danielson K.G. Iozzo R .V. LaMarca M. McKinney C.E. Ginns E.I. Bornstein P. J. Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (398) Google Scholar, 4Kyriakides T.R. Leach K.J. Hoffman A.S. Ratner B.D. Bornstein P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4449-4454Crossref PubMed Scopus (136) Google Scholar, 5Kyriakides T.R. Tam J.W.Y. Bornstein P. J. Invest. Dermatol. 1999; 113: 782-787Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Dermal fibroblasts, isolated from adult animals, also show an adhesive defect in vitro that is most evident when cells are plated on a variety of pure protein substrates in the absence of serum. Adhesion was restored by prolonged (48 h) incubation of TSP2-null cells with recombinant mouse TSP2 or by transfection with a TSP2 cDNA gene (6Yang Z. Kyriakides T.R. Bornstein P. Mol. Biol. Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar). The basis for this adhesive defect was recently investigated and was shown by zymography to result from an increase in matrix metalloproteinase 2 (MMP2) levels in both the conditioned media and cell layers of cultured cells (6Yang Z. Kyriakides T.R. Bornstein P. Mol. Biol. Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar). Although virtually all of the enzyme that was analyzed was in the zymogen or pro-MMP2 form, an increase in active MMP2 in TSP2-null cells was inferred from the observation that inhibitors of MMP2, including TIMP2 and a neutralizing anti-MMP2 antibody, corrected the adhesive defect (6Yang Z. Kyriakides T.R. Bornstein P. Mol. Biol. Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar).Both TSP2 and its close relative, TSP1, are known to interact with the scavenger receptor, low density lipoprotein-related receptor protein (LRP), an interaction that results in the endocytosis and lysosomal degradation of the TSP (7Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (127) Google Scholar, 8Chen H. Strickland D.K. Mosher D.F. J. Biol. Chem. 1996; 271: 15993-15999Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 9Chen H. Sottile J. Strickland D.K. Mosher D.F. Biochem. J. 1996; 318: 959-963Crossref PubMed Scopus (54) Google Scholar, 10Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This interaction is mediated by the NH2-terminal heparin-binding domain of the TSP and is competed by heparin (7Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (127) Google Scholar, 9Chen H. Sottile J. Strickland D.K. Mosher D.F. Biochem. J. 1996; 318: 959-963Crossref PubMed Scopus (54) Google Scholar, 10Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TSP1 and TSP2 also interact with a number of serine proteases, including plasmin, cathepsin G, and neutrophil elastase, and function as competitive inhibitors of these enzymes (12Hogg P.J. Thromb. Homeostasis. 1994; 72: 787-792Crossref PubMed Scopus (63) Google Scholar). In view of the fact that LRP is capable of binding and endocytosing both α2-macroglobulin proteinase and plasminogen activator inhibitor 1-tissue plasminogen activator and -urokinase-type plasminogen activator complexes (13Strickland D.K. Kounnas M.Z. Argraves W.S. FASEB J. 1995; 9: 890-898Crossref PubMed Scopus (248) Google Scholar), we postulated that TSP2-MMP2 complexes might also be cleared by the scavenger receptor.In this study we find that although TSP2 binds both pro-MMP2 and MMP2 directly, the protein does not function as a direct binding inhibitor of the active protease nor does it prevent the activation of pro-MMP2. However, TSP2-null cells were defective in the uptake of extracellular MMP2. Furthermore, inhibitors of LRP function reduced adhesion of control skin fibroblasts and, correspondingly, increased MMP2 levels in these cells. We therefore propose that the interaction of MMP2 with TSP2, and possibly also with TSP1, and the subsequent uptake of the protein-enzyme complex by LRP serve as a means of regulating extracellular MMP2 levels. These results have implications not only for the phenotype of the TSP2-null mouse but also for the control of processes such as collagen fibrillogenesis, wound healing, and angiogenesis. Thrombospondins (TSP)1 1 and 2 are large extracellular macromolecules whose diverse functions reflect their ability to bind to multiple cell-surface receptors, cytokines, growth factors, and proteases, and to structural components of the matrix (1Lawler J. Curr. Opin. Cell Biol. 2000; 12: 634-640Crossref PubMed Scopus (372) Google Scholar, 2Bornstein P. Armstrong L.C. Hankenson K.D. Kyriakides T.R. Yang Z. Matrix Biol. 2000; 19: 557-568Crossref PubMed Scopus (135) Google Scholar). TSP2-null mice display a complex phenotype that is characterized by changes in connective tissues, particularly in response to injury, an increase in vascular density and endosteal bone growth, and a bleeding defect (3Kyriakides T.R. Zhu Y.-H. Smith L.T. Bain S.D. Yang Z. Lin M.T. Danielson K.G. Iozzo R .V. LaMarca M. McKinney C.E. Ginns E.I. Bornstein P. J. Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (398) Google Scholar, 4Kyriakides T.R. Leach K.J. Hoffman A.S. Ratner B.D. Bornstein P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4449-4454Crossref PubMed Scopus (136) Google Scholar, 5Kyriakides T.R. Tam J.W.Y. Bornstein P. J. Invest. Dermatol. 1999; 113: 782-787Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Dermal fibroblasts, isolated from adult animals, also show an adhesive defect in vitro that is most evident when cells are plated on a variety of pure protein substrates in the absence of serum. Adhesion was restored by prolonged (48 h) incubation of TSP2-null cells with recombinant mouse TSP2 or by transfection with a TSP2 cDNA gene (6Yang Z. Kyriakides T.R. Bornstein P. Mol. Biol. Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar). The basis for this adhesive defect was recently investigated and was shown by zymography to result from an increase in matrix metalloproteinase 2 (MMP2) levels in both the conditioned media and cell layers of cultured cells (6Yang Z. Kyriakides T.R. Bornstein P. Mol. Biol. Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar). Although virtually all of the enzyme that was analyzed was in the zymogen or pro-MMP2 form, an increase in active MMP2 in TSP2-null cells was inferred from the observation that inhibitors of MMP2, including TIMP2 and a neutralizing anti-MMP2 antibody, corrected the adhesive defect (6Yang Z. Kyriakides T.R. Bornstein P. Mol. Biol. Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar). Both TSP2 and its close relative, TSP1, are known to interact with the scavenger receptor, low density lipoprotein-related receptor protein (LRP), an interaction that results in the endocytosis and lysosomal degradation of the TSP (7Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (127) Google Scholar, 8Chen H. Strickland D.K. Mosher D.F. J. Biol. Chem. 1996; 271: 15993-15999Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 9Chen H. Sottile J. Strickland D.K. Mosher D.F. Biochem. J. 1996; 318: 959-963Crossref PubMed Scopus (54) Google Scholar, 10Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This interaction is mediated by the NH2-terminal heparin-binding domain of the TSP and is competed by heparin (7Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (127) Google Scholar, 9Chen H. Sottile J. Strickland D.K. Mosher D.F. Biochem. J. 1996; 318: 959-963Crossref PubMed Scopus (54) Google Scholar, 10Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TSP1 and TSP2 also interact with a number of serine proteases, including plasmin, cathepsin G, and neutrophil elastase, and function as competitive inhibitors of these enzymes (12Hogg P.J. Thromb. Homeostasis. 1994; 72: 787-792Crossref PubMed Scopus (63) Google Scholar). In view of the fact that LRP is capable of binding and endocytosing both α2-macroglobulin proteinase and plasminogen activator inhibitor 1-tissue plasminogen activator and -urokinase-type plasminogen activator complexes (13Strickland D.K. Kounnas M.Z. Argraves W.S. FASEB J. 1995; 9: 890-898Crossref PubMed Scopus (248) Google Scholar), we postulated that TSP2-MMP2 complexes might also be cleared by the scavenger receptor. In this study we find that although TSP2 binds both pro-MMP2 and MMP2 directly, the protein does not function as a direct binding inhibitor of the active protease nor does it prevent the activation of pro-MMP2. However, TSP2-null cells were defective in the uptake of extracellular MMP2. Furthermore, inhibitors of LRP function reduced adhesion of control skin fibroblasts and, correspondingly, increased MMP2 levels in these cells. We therefore propose that the interaction of MMP2 with TSP2, and possibly also with TSP1, and the subsequent uptake of the protein-enzyme complex by LRP serve as a means of regulating extracellular MMP2 levels. These results have implications not only for the phenotype of the TSP2-null mouse but also for the control of processes such as collagen fibrillogenesis, wound healing, and angiogenesis. We thank members of our laboratories for helpful discussions and a careful reading of the manuscript.