Heparin Potentiates the in Vivo Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Abstract

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists. Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists. Bone morphogenetic proteins (BMPs) 2The abbreviations used are: BMPs, bone morphogenetic proteins; TGF-β, transforming growth factor-β; ALP, alkaline phosphatase.2The abbreviations used are: BMPs, bone morphogenetic proteins; TGF-β, transforming growth factor-β; ALP, alkaline phosphatase. were originally identified as an activity inducing ectopic bone formation when implanted into muscle tissue (1Urist M.R. Science. 1965; 150: 893-899Crossref PubMed Scopus (4426) Google Scholar). Isolation of BMPs using heparin affinity column chromatography and molecular cloning of BMPs revealed that the ectopic bone-inducing activity is due to several BMPs (2Sampath T.K. Muthukumaran N. Reddi A.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7109-7113Crossref PubMed Scopus (289) Google Scholar, 3Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3306) Google Scholar). More than 15 BMPs have since been identified in vertebrates and are classified into several subgroups: the BMP-2/4 subgroup, the BMP-5/6/7/8 subgroup, the GDF (growth and differentiation factor)-5 (also termed BMP-14)/GDF-6 (BMP-13)/GDF-7 (BMP-12) subgroup, and the BMP-3 subgroup (4Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (447) Google Scholar, 5Canalis E. Economides A.N. Gazzerro E. Endocr. Rev. 2003; 24: 218-235Crossref PubMed Scopus (715) Google Scholar, 6Waite K.A. Eng C. Nat. Rev. Genet. 2003; 4: 763-773Crossref PubMed Scopus (226) Google Scholar). With the exception of BMP-1, BMPs are members of the TGF-β superfamily, which includes multifunctional factors in vertebrates and invertebrates (3Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3306) Google Scholar). BMPs play critical roles in skeletal development, especially in mammals (4Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (447) Google Scholar, 5Canalis E. Economides A.N. Gazzerro E. Endocr. Rev. 2003; 24: 218-235Crossref PubMed Scopus (715) Google Scholar, 6Waite K.A. Eng C. Nat. Rev. Genet. 2003; 4: 763-773Crossref PubMed Scopus (226) Google Scholar, 7Wan M. Cao X. Biochem. Biophys. Res. Commun. 2005; 328: 651-657Crossref PubMed Scopus (310) Google Scholar). BMPs regulate the differentiation and function of cells that are involved in bone and cartilage formation and deformation, including osteoblasts, chondrocytes, and osteoclasts (4Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (447) Google Scholar, 5Canalis E. Economides A.N. Gazzerro E. Endocr. Rev. 2003; 24: 218-235Crossref PubMed Scopus (715) Google Scholar, 7Wan M. Cao X. Biochem. Biophys. Res. Commun. 2005; 328: 651-657Crossref PubMed Scopus (310) Google Scholar). A cell culture system using C2C12 myoblasts can mimic part of the process of the ectopic bone formation induced by BMPs in muscle tissue (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). BMPs inhibit myogenic differentiation of myoblasts into mature muscle cells and convert their differentiation pathway into that of osteoblast lineage cells. Using this model system in vitro, a critical factor for osteoblast differentiation (termed Osterix) has been cloned as a novel zinc finger transcription factor induced by BMP-2 (9Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2700) Google Scholar). Osterix knock-out mice do not have bone tissues because of lack of osteoblast differentiation from mesenchymal progenitor cells (9Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2700) Google Scholar). Although several growth factors, including TGF-β and activin, inhibit the myogenic differentiation of C2C12 cells in vitro, they do not induce osteoblast differentiation (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar, 10Ladd A.N. Yatskievych T.A. Antin P.B. Dev. Biol. 1998; 204: 407-419Crossref PubMed Scopus (146) Google Scholar). It has been reported that TGF-β itself fails to induce ectopic bone formation in vivo (2Sampath T.K. Muthukumaran N. Reddi A.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7109-7113Crossref PubMed Scopus (289) Google Scholar). These findings suggest that the C2C12 cell model system is useful for examining the molecular mechanisms of TGF-β superfamily members during ectopic bone formation in muscle tissue. It should therefore be useful for examining the molecular mechanism of bone formation and osteoblast differentiation induced by BMPs. BMP signaling is transduced by two types of transmembrane serine/threonine kinase receptors. BMP receptors IA (ALK-3) and IB (ALK-6), ALK-1, and ALK-2 function as type I BMP receptors, whereas BMP receptor II and activin receptors II and IIB function as type II receptors, which are constitutively active kinases (4Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (447) Google Scholar, 11Yoon B.S. Lyons K.M. J. Cell. Biochem. 2004; 93: 93-103Crossref PubMed Scopus (245) Google Scholar, 12Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Crossref PubMed Scopus (574) Google Scholar, 13von Bubnoff A. Cho K.W. Dev. Biol. 2001; 239: 1-14Crossref PubMed Scopus (336) Google Scholar). After type II receptors phosphorylate type I receptors in a ligand-dependent fashion, activated type I receptors phosphorylate downstream molecules in the cytoplasm. The Smad1/5/8 transcription factors are phosphorylated by BMP type I receptors in the cytoplasm as substrates and accumulate in the nucleus within 1 h after BMP stimulation (4Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (447) Google Scholar, 11Yoon B.S. Lyons K.M. J. Cell. Biochem. 2004; 93: 93-103Crossref PubMed Scopus (245) Google Scholar, 12Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Crossref PubMed Scopus (574) Google Scholar, 13von Bubnoff A. Cho K.W. Dev. Biol. 2001; 239: 1-14Crossref PubMed Scopus (336) Google Scholar). The phosphorylated Smad proteins directly regulate expression of primary target genes through binding to their promoter or enhancer elements together with Smad4 and other transcription factors (14ten Dijke P. Korchynskyi O. Valdimarsdottir G. Goumans M.J. Mol. Cell. Endocrinol. 2003; 211: 105-113Crossref PubMed Scopus (171) Google Scholar, 15Ito Y. Miyazono K. Curr. Opin. Genet. Dev. 2003; 13: 43-47Crossref PubMed Scopus (266) Google Scholar). BMP activity is positively and negatively regulated by many types of molecules both intracellularly and in the extracellular environment (7Wan M. Cao X. Biochem. Biophys. Res. Commun. 2005; 328: 651-657Crossref PubMed Scopus (310) Google Scholar, 13von Bubnoff A. Cho K.W. Dev. Biol. 2001; 239: 1-14Crossref PubMed Scopus (336) Google Scholar, 16Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). Various corepressors and coactivators of Smad proteins have been identified in the cytoplasm or nucleus in mammalian cells. Noggin, chordin, follistatin, and the DAN family proteins bind to BMPs in the extracellular space and inhibit binding of ligands to cell-surface receptors (16Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). In contrast, heparin enhances BMP-induced osteoblast differentiation in C2C12 myoblasts in vitro (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Other types of sulfated polysaccharides such as native heparan sulfate and synthetic dextran sulfate also stimulate BMP-2 (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The capacity of these sulfated polysaccharides to stimulate BMP-2 appears to depend on the size and number of their sulfated residues (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Heparin does not enhance osteoblast differentiation induced by direct activation of the BMP intracellular signaling pathway by overexpression of a constitutively active BMP receptor without addition of BMPs (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). This finding that heparin of the BMP including BMP than on the BMP intracellular signaling Heparin has been reported to enhance the of growth factors by the affinity of ligands for receptors as a B. N. Y. Acad. Sci. PubMed Scopus Google Scholar, A. M. P. Cell. Full Text PDF PubMed Scopus Google Scholar). heparin the of in C2C12 that a novel mechanism of the heparin activity is involved in the of BMPs (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The and capacity of BMPs to induce new bone formation in vivo should be useful for the of for in vivo bone (1Urist M.R. Science. 1965; 150: 893-899Crossref PubMed Scopus (4426) Google Scholar, N. J. Oral Sci. 2003; PubMed Scopus Google Scholar). has been reported that larger amounts of BMPs are required to induce bone formation in such as monkeys and than in K. K. K. J. Y. Wang Y. T. Y. J. Res. 2002; PubMed Scopus Google Scholar). We that simultaneous administration of BMPs with sulfated polysaccharides the amounts of BMPs required to induce clinically useful amounts of new bone in vivo. In this study, we found that heparin stimulates the ectopic activity of BMP-2 in vivo and a novel mechanism of the of heparin on BMP signaling in target cells. and BMP-2 was from and and were from Heparin from and molecular heparin was from heparin for into was from Cell myoblasts were and with BMPs as (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar, T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). cells were at a of 1 The cells were with BMPs in the presence or of heparin in medium The of BMP-2 in the culture medium were using an activity was as a of osteoblast differentiation (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). with an were with a of 1 and The was by and A were at gene expression were by T. Katagiri T. S. M. T. Takahashi N. Suda T. J. Bone 2001; PubMed Scopus Google Scholar). were from C2C12 cells with and using II was with and the ALP, and and type and types II and and and and and and was at for by at for at for 1 and at for 1 determine cells were with primary and as (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). The primary were sulfate Smad1/5/8 and determine activity cells were for activity as (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). was with a with a cells were with BMP-2 in the presence or of heparin and in and and as (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, T. S. M. Komaki M. Yamaguchi A. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. Cell Res. PubMed Scopus Google Scholar). The were to and to and Smad1/5/8 proteins were using Smad1/5/8 and of the of cells were for to h with BMP-2 in medium the culture medium and cell which were by the cells with 1 for at were and at The amounts of BMP-2 in the culture medium and cell were using the to the Bone of heparin on bone formation in vivo induced by BMP-2 were examined by ectopic bone formation in mice R. K. K. Bone 2005; PubMed Scopus Google Scholar). of BMP-2 and or of heparin were and to of a from and at implanted into were mice were and in with to and for 1 and they were into The mice were by and the as were implanted into both muscle of the and after the mice were and the were and for as were with a The bone of was by using a bone of the or tissue from were with and examined a in bone were by of for when of a was used to which for the The were using of the of Heparin on of by compared the of heparin on a of osteoblast induced by and in C2C12 cells. we reported (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google heparin at enhanced the activity induced by both BMP-2 and A and In contrast, the activity induced by was by heparin a of mammalian BMP-2 and failed to induce activity in the presence or of heparin in system not although heparin on BMP-2 to of the heparin used this study molecular heparin, which is than native heparin, also enhanced of activity by but to a than native heparin The of molecular heparin and native heparin were and that the size of sulfated polysaccharides is critical for of of activity by BMPs. of and with Heparin in to cells heparan sulfate on the cell in the extracellular space We examined the of heparan sulfate in to BMP-2. C2C12 cells were with and with BMP-2 in the presence of heparan sulfate was from the in a of cells were induced by BMP-2 regardless of the presence of Furthermore, the of the of BMP-2 on activity was nearly in the presence and of We reported (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google that heparin does not enhance osteoblast differentiation induced by activation of a signaling pathway downstream of BMP receptors, that a primary target of heparin is of the In with this of C2C12 cells with heparin on to did not enhance the activity induced by BMP-2 on of BMP-2 with heparin treatment did not affect activity Although we the that the with heparin was on these suggest that heparin BMP-2 in culture of the of Heparin on BMP-2 examined the of in the expression of genes to osteoblast such as ALP, and type I and types II and The of type I were not by BMP stimulation with or without heparin h. of the other genes was induced by BMP-2 and by heparin after 24 h. the of of Osterix by BMP-2 at h was not enhanced by heparin We whether heparin the of phosphorylation of Smad1/5/8 induced by BMP-2. BMP-2-induced phosphorylation of Smad1/5/8 within 1 h and h A and the phosphorylation of Smad proteins were not within 12 h by heparin, but were at 24 and h This of heparin on the phosphorylation of Smad proteins was by of phosphorylated Smad1/5/8 in was by BMP-2 stimulation at 24 h and only in the presence of heparin We examined the of the of heparin on the phosphorylation of Smad proteins induced by BMP-2. C2C12 cells were with BMP-2 in the presence or of heparin for 1 or 24 h on 1 or after cell to the of cell growth on the capacity of heparin. Smad proteins were only in for 24 h with BMP-2 in the presence of heparin that the of Smad1/5/8 phosphorylation by heparin on the of treatment and not the of We examined the of heparin on the of phosphorylated Smad C2C12 cells were for 1 or h with BMP-2 to induce Smad without and examined for the of phosphorylated Smad proteins at 24 h. Smad proteins were in with BMP-2 for 1 or h in the presence of heparin that heparin enhances the of phosphorylated Smad proteins induced by BMP-2 without their Heparin the of BMP-2 in phosphorylated Smad proteins were by heparin only in the presence of we the amounts of BMP-2 in the culture medium in the presence and of heparin. in the of heparin, the of BMP-2 in the culture medium of the within 1 h and was after h In contrast, in the presence of heparin, of BMP-2 in the culture medium at 12 and BMP-2 was at h We also the amounts of BMP-2 in cell the culture in the amounts of BMP-2 were in the presence and of that the of BMP-2 from the culture medium is due to degradation of the than in cell We examined whether BMP-2 in culture medium the differentiation of C2C12 cells in the of heparin. C2C12 cells were for with of myogenic differentiation was by and osteoblast differentiation was induced by BMP-2 (8Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). these were to to when the culture medium was 24 h to the of BMP-2 in the medium These findings suggest that heparin the in the BMP in culture medium and that of BMP-2 are of the pathway of differentiation of C2C12 cells they are by of Heparin BMP from by a BMP that BMP signaling in the extracellular is induced by BMPs as an gene E. V. E. J. 1998; PubMed Scopus Google Scholar). mRNA was induced within 1 h after BMP-2 stimulation in C2C12 cells The of of noggin mRNA by BMP-2 was not enhanced by heparin at 1 but was at h Furthermore, noggin dose-dependently the of cells induced by BMP-2. In contrast, BMP-2 with heparin induced activity compared with BMP-2 alone. this large of cells in the presence of noggin These suggest that heparin BMPs from inhibition by Heparin the Bone by BMP-2 in we examined the of heparin on BMP-2 activity in vivo. BMP-2 induced ectopic bone formation within in the size of new bone induced by BMP-2 for to A and Heparin bone tissue induced by BMP-2 after of than did BMP-2 not Bone was also by heparin in a the of the bone with BMP-2 were with bone In the presence of heparin, not only bone but also a large of bone was in the including have reported that sulfated polysaccharides such as heparin, heparan and dextran sulfate enhance the osteoblast differentiation induced by BMPs in vitro (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, R. E. W. J. Biochem. PubMed Scopus Google Scholar). In this study, we have that heparin enhanced the bone formation induced by BMP-2 in vivo as In the presence of heparin, BMP-2 induced larger amounts of mineralized bone tissues within in we have that molecular heparin, which is than native heparin and is used also enhanced BMP-2 activity at in vitro, although the activity of molecular heparin in this was than that of native heparin. This finding that sulfated polysaccharides stimulate BMP in a (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). these findings suggest that simultaneous of BMP-2 and sulfated polysaccharides is useful for of bone tissues in vivo. This in in vivo of bone in cells types of which of proteins and including heparan sulfate M. T. C. T. T.A. M. M. E. J. Bone 2005; PubMed Scopus Google Scholar, Nat. Rev. Mol. Cell Biol. 2003; 4: PubMed Scopus Google Scholar, M. Biochem. Sci. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We have that not only heparin but also other types of sulfated polysaccharides such as native heparan sulfate and synthetic dextran sulfate enhance BMP (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). These accumulate on the cell and in the extracellular and regulate BMP has been reported that cell-surface inhibit the activity of in cells A. K. Y. Biochem. Biophys. Res. Commun. 2003; PubMed Scopus Google Scholar). of heparan sulfate from of C2C12 cells did not affect the osteoblast differentiation that in response to BMP-2. This in findings due to in the cell types or in BMP BMP-2. heparin the activity that was by in C2C12 cells. is classified into the subgroup as not on A.J. Hewick R.M. Rosen V. Wang E.A. Wozney J.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). It appears that the or of sulfated polysaccharides on BMP depend on the primary of BMPs. Although of were identified in the bone-inducing from bone using a heparin the of the of BMP-2 and which are to be the are from of and R. E. W. J. Biochem. PubMed Scopus Google Scholar, A.J. Hewick R.M. Rosen V. Wang E.A. Wozney J.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). These affect the and of types of receptors have been identified for the BMP-2 and (4Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (447) Google Scholar, 5Canalis E. Economides A.N. Gazzerro E. Endocr. Rev. 2003; 24: 218-235Crossref PubMed Scopus (715) Google Scholar). These Heparin is to the binding affinity of growth factor for receptor and enhance downstream signaling B. N. Y. Acad. Sci. PubMed Scopus Google Scholar, A. M. P. Cell. Full Text PDF PubMed Scopus Google Scholar). A mechanism to in the of BMPs because heparin does not stimulate formation BMP-2 and the BMP type IA receptor on the of C2C12 cells (17Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). heparin failed to stimulate in response to such as the phosphorylation of Smad proteins and the of including noggin and heparin required h to stimulate the induced by BMP-2. we found that heparin the of BMP-2 in the culture medium at and BMP-2 from inhibition by which was induced as part of the in response to BMP-2. the of the findings we we a novel mechanism for the activity of heparin on BMPs In the of heparin, BMP are negatively regulated by the in the amounts of active ligands due to and BMP are induced by BMP signaling as part of the to in the presence of heparin, BMPs in the extracellular space for a and active ligands are from by antagonists. In heparin not only the osteoblast differentiation induced by BMP-2 and in vitro but also the bone formation induced by BMP-2 in vivo. It appears that heparin stimulates BMP by protecting ligands from the target cells than These findings suggest that simultaneous administration of BMPs with sulfated polysaccharides should be clinically useful at for bone We members of the of of and the of for for and We also Miyazono and for for the and for BMP-2.