Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity
Sclerosteosis is a progressive sclerosing bone dysplasia. Sclerostin (the SOST gene) was originally identified as the sclerosteosis-causing gene. However, the physiological role of sclerostin remains to be elucidated. Sclerostin was intensely expressed in developing bones of mouse embryos. Punctuated expression of sclerostin was localized on the surfaces of both intramembranously forming skull bones and endochondrally forming long bones. Sclerostin-positive cells were identified as osteoclasts. Recombinant sclerostin protein produced in cultured cells was efficiently secreted as a monomer. We examined effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for mouse preosteoblastic MC3T3-E1 cells. Sclerostin inhibited the BMP6 and BMP7 activity but not the BMP2 and BMP4 activity. Sclerostin bound to BMP6 and BMP7 with high affinity but bound to BMP2 and BMP4 with lower affinity. In conclusion, sclerostin is a novel secreted osteoclast-derived BMP antagonist with unique ligand specificity. We suggest that sclerostin negatively regulates the formation of bone by repressing the differentiation and/or function of osteoblasts induced by BMPs. Since sclerostin expression is confined to the bone-resorbing osteoclast, it provides a mechanism whereby bone apposition is inhibited in the vicinity of resorption. Our findings indicate that sclerostin plays an important role in bone remodeling and links bone resorption and bone apposition. Sclerosteosis is a progressive sclerosing bone dysplasia. Sclerostin (the SOST gene) was originally identified as the sclerosteosis-causing gene. However, the physiological role of sclerostin remains to be elucidated. Sclerostin was intensely expressed in developing bones of mouse embryos. Punctuated expression of sclerostin was localized on the surfaces of both intramembranously forming skull bones and endochondrally forming long bones. Sclerostin-positive cells were identified as osteoclasts. Recombinant sclerostin protein produced in cultured cells was efficiently secreted as a monomer. We examined effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for mouse preosteoblastic MC3T3-E1 cells. Sclerostin inhibited the BMP6 and BMP7 activity but not the BMP2 and BMP4 activity. Sclerostin bound to BMP6 and BMP7 with high affinity but bound to BMP2 and BMP4 with lower affinity. In conclusion, sclerostin is a novel secreted osteoclast-derived BMP antagonist with unique ligand specificity. We suggest that sclerostin negatively regulates the formation of bone by repressing the differentiation and/or function of osteoblasts induced by BMPs. Since sclerostin expression is confined to the bone-resorbing osteoclast, it provides a mechanism whereby bone apposition is inhibited in the vicinity of resorption. Our findings indicate that sclerostin plays an important role in bone remodeling and links bone resorption and bone apposition. Sclerosteosis is a progressive sclerosing bone dysplasia with an autosomal recessive mode of inheritance. Scleroteosis is clinically and radiologically very similar to van Buchem disease (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). By linkage analysis of families with these diseases, the disease-causing genes were mapped to the same chromosomal 17q12-q21 region, supporting the hypothesis that both diseases are caused by mutations in the same gene. By the positional cloning strategy, sclerostin (the SOST gene), which was mutated in sclerosteosis patients, was identified (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). Sclerostin was found to be expressed in human long bones and cartilage using the polymerase chain reaction. However, the expression of sclerostin in the bones and cartilage was not examined in detail. The pathogenesis and genetics of sclerosteosis suggest that inhibition of sclerostin could lead to increased bone density. This definitely makes sclerostin and its pathway interesting targets for the development of anabolic agents against osteoporosis (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). Sclerostin encodes a protein of 213 amino acids with a putative signal peptide for secretion, and sclerostin has six conserved cysteine residues and one conserved glycine residue that are essential to form a cystine knot. The spacing of cysteine residues is highly homologous to that of bone morphogenetic protein (BMP) 1The abbreviations used are: BMP, bone morphogenetic protein; En, embryonic day n. antagonists of the DAN/cerberus family, indicating that sclerostin might be a BMP antagonist (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). However, the biological activity of sclerostin is not known. Therefore, the physiological role of sclerostin and its mechanism of action remain to be elucidated. We examined the expression of sclerostin in mouse embryonic bones by in situ hybridization and the biological activity of recombinant sclerostin protein. Sclerostin was found to be expressed in osteoclasts both in bones forming directly from mesenchyme and via a cartilage template. We showed that sclerostin encoded a novel secreted osteoclast-derived BMP antagonist with unique ligand specificity. The present findings indicate that sclerostin negatively regulates the formation of bone by repressing the differentiation and/or function of osteoblasts induced by BMPs. Sclerostin apparently links the functions of osteoclasts and osteoblasts in bone resorption and bone apposition, respectively, and it therefore provides a novel mechanism for the regulation of bone remodeling. In Situ Hybridization—The embryonic mouse tissues (embryonic day 10 to newborn) were fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned. Radioactive in situ hybridization procedures for tissue sections were carried out as described (3Wilkinson D. Green J. Copp A.J. Cole D.E. Postimplantation Mammalian Embryos. Oxford University Press, London1990: 155-171Google Scholar). Probes were labeled with [35S]UTP; exposure time was 14 or 28 days. The preparation of the Bsp and MMP-9 RNA probes have been previously described (4Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Google Scholar, 5Rice D.P.C. Kim H.-J. Thesleff I. Eur. J. Oral Sci. 1999; 107: 265-275Google Scholar). The sclerostin RNA probe was prepared using mouse sclerostin (SOST) cDNA (680 bp) as a template (2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). Production of Recombinant Mouse Sclerostin in Insect Cells—The mouse sclerostin cDNA with a 75-base pair DNA fragment encoding an E tag (GAPVPYPDPLEPR) and a His6 tag (HHHHHH) at the 3′ terminus of the coding region was constructed in a transfer vector DNA, pBacPAK9. Recombinant baculovirus containing the sclerostin cDNA with the tag sequences was obtained by cotransfection of Sf9 cells with the recombinant pBacPAK9 and a Bsu36I-digested expression vector, BacPak6. High Five cells infected with the recombinant baculovirus were cultured at 27 °C for 72 h in Grace's insect cell culture medium (Invitrogen) containing 10% fetal bovine serum. Recombinant mouse sclerostin was purified from the culture medium by affinity chromatography using nickel-nitrilotriacetic acid-agarose and desalted by gel filtration chromatography using Bio-Gel P-6 DG in PBS containing 50 μg/ml bovine serum albumin. Detection of Recombinant Mouse Sclerostin by Western Blotting Analysis—The culture medium and cell lysate of High Five cells infected with recombinant baculovirus were separated by SDS-12.5% polyacrylamide gel electrophoresis under reducing or nonreducing conditions and transferred onto a nitrocellulose membrane (Hybond-ECL). The protein with the E tag on the membrane was visualized as described (6Miyake M. K. S. T. T. N. Scholar). recombinant mouse sclerostin protein was separated by SDS-12.5% polyacrylamide gel electrophoresis under reducing conditions and with was used as MC3T3-E1 Cell preosteoblastic MC3T3-E1 cells were and for or at °C in a of in in essential medium containing 10% fetal bovine and μg/ml in a in MC3T3-E1 of MC3T3-E1 cells were at a of in the cells the medium was with essential medium containing μg/ml 10 and 50 μg/ml and cells were cultured for The cells were cultured in essential medium containing fetal bovine μg/ml 10 50 μg/ml 10 recombinant human BMP2 BMP4 BMP6 or BMP7 protein and recombinant mouse sclerostin protein or recombinant mouse for 72 were with PBS and in 10 and The cell were on of were for protein and activity as described Y. M. T. E. T. T. J. Biol. Scholar). the the 10 in with and was at °C for was the of was using a of Sclerostin to the Recombinant mouse sclerostin protein was fixed on a The of BMP2, BMP4, BMP6, or BMP7 to the sclerostin on the was using the The was using the of Sclerostin sclerosteosis bone and remodeling in the skull (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google examined the expression of sclerostin in the developing skull of mouse by in situ skull bones directly from the mesenchyme by the mechanism of bone The of bones is in embryonic day mouse embryos. However, sclerostin expression was in the mesenchyme in sections of and not cells sclerostin were at at the of not Sclerostin expression increased at and it as expression in the developing and bones The sclerostin cells were localized on the surfaces of forming but of the developing bones. This is by the expression of the bone which the of bone formation In sclerostin cells were in developing bones in the cells sclerostin were present on the surfaces of the forming bones and in the bone was in the bone the Since sclerosteosis bone and remodeling in the region of long bones (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google examined the expression of sclerostin in developing long bones and bones of mouse by in situ In the at sclerostin was in the of the region and the of the region In the at sclerostin was in the bone in to the and We examined the expression of sclerostin in endochondrally developing bones the and The expression in of these bones was similar to that in the not In sclerostin cells were the cartilage Sclerostin expression was to the and tissues in the sections of were expression was found in the of the in In expression was in the of and it was localized to of cells of Sclerostin with MMP-9 the sclerostin cells were with osteoblasts on the surfaces of forming was to of bone Sclerostin expression was and it to to cells. This that of bone-resorbing osteoclasts. The MMP-9 has been to be a of in in the developing bones (4Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Google Scholar, D.P.C. Kim Thesleff I. Scholar). of the of MMP-9 and sclerostin cells in the intramembranously forming bones in the embryonic showed that the were similar This that sclerostin is expressed in osteoclasts. The of sclerostin in the long bones is similar to that of MMP-9 (4Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Google but not to that of the a of osteoblasts S. S. Biol. Scholar). sclerostin is expressed in osteoclasts in the long bones. Production of Recombinant Mouse is a putative secreted protein with a putative secreted signal peptide (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). sclerostin is a secreted mouse sclerostin cDNA was expressed in cultured High Five insect cells by with recombinant baculovirus containing the mouse sclerostin cDNA with the encoding E and His6 recombinant mouse both the culture medium and cell lysate were examined by reducing gel electrophoresis by Western analysis with tag with similar of were in the culture medium but not in the cell that sclerostin is a secreted protein The were the of recombinant sclerostin Since sclerostin has at and (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google it might be The of sclerostin might from the of We examined mouse recombinant sclerostin by nonreducing gel electrophoresis by Western Sclerostin as a indicating that it was secreted as a Recombinant mouse sclerostin was purified from the culture medium by affinity chromatography using nickel-nitrilotriacetic acid-agarose by gel recombinant sclerostin was examined by reducing gel electrophoresis by protein sclerostin showed of of Sclerostin on BMP in is a of the cystine The spacing of cysteine residues in sclerostin is highly homologous to that of BMP and of the DAN/cerberus family, indicating that sclerostin might be a BMP antagonist (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). were originally identified as in bone that bone and cartilage formation in are for the of differentiation Genet. 1994; Scholar, 10: Scholar). We examined effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for the differentiation of MC3T3-E1 cells by a for differentiation Y. J. 1994; Scholar). BMP2, BMP4, BMP6, and BMP7 the activity in MC3T3-E1 cells We examined effects of recombinant sclerostin protein on the BMP activity. The activity of BMP6 was inhibited by sclerostin in a The activity of BMP7 was but inhibited by sclerostin In the activity of BMP2 and BMP4 was not inhibited by sclerostin and a BMP is to BMP2, BMP4, and with a activity for BMP2 and BMP4 Scholar). We examined the of recombinant mouse protein on the BMP activity. In to the activity of BMP2, BMP4, BMP6, and with a activity for BMP2 and BMP4 of Sclerostin to sclerostin inhibited the activity of BMP6, examined the of sclerostin to BMP6 using the with the recombinant sclerostin and of BMP6 were obtained The was Sclerostin was found to to BMP6 with high affinity We examined the of sclerostin to BMP2, BMP4, and and were obtained not sclerostin was found to to BMP7 with lower affinity and to BMP2 and BMP4 with lower affinity of sclerostin to in a Sclerosteosis is a progressive sclerosing bone dysplasia by bone with an autosomal recessive mode of inheritance. The bone and in the skull and region of long bones (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). activity and increased of bone formation have a in Sclerostin (the SOST gene) was originally identified by positional cloning of the disease-causing in Therefore, inhibition of sclerostin was to lead to increased bone definitely sclerostin and its pathway interesting targets for the development of anabolic agents against osteoporosis (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). However, the physiological role of sclerostin and its mechanism of action remain to be elucidated. are and B.R. W. Cell Biol. Scholar, P.J. Scholar). of skull bones is by the of the long bones and the of in which cartilage is Since both skull bones and long bones are in the sclerosteosis patients, sclerostin is apparently in both and bone formation (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). Sclerostin was expressed on the surfaces of forming bones in the embryonic mouse The bones are as of cells. The cells directly to osteoblasts and the of which The development is by the of bone remodeling the of the bone is by localized bone resorption by osteoclasts and apposition by in the skull sclerostin was expressed in the long bones. is by of cells. are that and the cartilage is by In a bone is via an mechanism by osteoblasts from cells in the B.R. W. Cell Biol. Scholar, P.J. Scholar). Sclerostin is expressed in the of the region as as the and bone of the This that sclerostin was expressed at the of bone formation in developing long bones. Sclerostin was not expressed in of the for the of the indicate that sclerostin plays a role in but not in in the development of long bones. Sclerostin was from the tissue in the as as are cells from the and a of the bone and the the expression of sclerostin was confined to the osteoclasts but not to We identified the osteoclasts in forming bones by the of the expression of a that is expressed in osteoclasts (4Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Google Scholar). for osteoclasts is the which is intensely expressed by the osteoclasts MMP-9 and expression in expression on the of bone D.P.C. Kim Thesleff I. Scholar). the osteoclasts bone are but the osteoclasts and bone bone development are and Since sclerostin was expressed very bone its expression with MMP-9 and showed that the genes were apparently localized to the same cells. We therefore that sclerostin is expressed in osteoclasts. sclerostin expression was in the embryonic in which are of osteoclasts. Since sclerostin has a putative signal at its amino it is to be a secreted protein (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). We expressed mouse sclerostin in cultured cells. Recombinant sclerostin protein was efficiently Sclerostin has six conserved cysteine residues and one conserved glycine residue that are essential to form the cystine (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). cystine are secreted as Biol. Scholar). The spacing of cysteine residues in sclerostin is highly homologous to that of BMP antagonists of the DAN/cerberus and of the DAN/cerberus family, are secreted as C. E. L. S. M. C. M. D. T. Biol. Scholar, E. C. S. L. S. T. B. C. M. M. D. T. Scholar). have an cysteine residue that is used for However, sclerostin not have cysteine Recombinant sclerostin was found to be secreted as a monomer. The of the spacing of cysteine residues in sclerostin to that in the DAN/cerberus that sclerostin might be a BMP antagonist (1Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 2Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J.T. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). are in are for the of differentiation Genet. 1994; Scholar, 10: Scholar). are to a for in mouse preosteoblastic MC3T3-E1 cells Y. J. 1994; Scholar). The BMP be on BMP2 and BMP4 form one BMP6, and form T. K. I. K. M. K. T. J. Cell Sci. 1999; Scholar). Therefore, examined the effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for the differentiation of MC3T3-E1 cells by the activity. Sclerostin was found to have activity for BMP6 and activity for BMP7 and for BMP2 and We examined the of sclerostin to these using the Sclerostin was found to to BMP6 with high affinity. In sclerostin bound to BMP7 with lower affinity and to BMP2 and BMP4 with lower affinity. were with of the inhibition Therefore, sclerostin to as a BMP antagonist by to a BMP was originally from an to development in by is to and BMP2, BMP4, and with a affinity for BMP2 and BMP4 Scholar). Our indicate that BMP2 and BMP4 and BMP6 and a BMP plays in the formation of the BMP2, BMP4, and BMP7 in a similar to S. Y. B. Scholar). and of the DAN/cerberus family, are to and BMP2, BMP4, and BMP7 Mol. Biol. 2001; Scholar, Y. C. 1999; Scholar, Mol. Scholar). indicate that sclerostin is a novel BMP antagonist with unique ligand specificity. were and but the of in a in confined to the developing E. Genet. Scholar). In situ hybridization have that BMP6 is expressed in with BMP genes in developing bones. it is that for BMP6 in the E. Genet. Scholar). In conclusion, sclerostin is a novel secreted BMP antagonist with unique ligand and its expression is confined to osteoclasts in both and bones. sclerostin is the BMP antagonist that is localized in osteoclasts. We suggest that sclerostin plays a role in bone development and that it negatively regulates the formation of bone by repressing the differentiation and/or function of Sclerostin to a mechanism whereby bone apposition is in the vicinity of bone resorption. Sclerostin links the of resorption and apposition via a previously The of osteoblasts and osteoclasts is to have an important role in the regulation of as by the regulation of differentiation by the ligand expressed by osteoblasts H. N. N. K. M. S. K. M. E. T. K. N. N. T. Sci. S. Scholar). The present findings a novel and in the of and functions bone remodeling.