Treatment with BMP-7 causes a shift in the differentiation pathway from myoblastic to osteoblastic in C2C12 mouse myoblast precursor cells in vitro. The underlying molecular mechanism is largely unknown. BMP-7 at 200 ng/ml completely inhibited myotube formation in C2C12 cells and dramatically induced alkaline phosphatase activity up to 20-fold when compared to untreated cells by day 12 in culture. The level of Runx2/Cbfa1 mRNA, a bone-specific transcription factor, was also stimulated up to 6-fold by BMP-7 with a peak at 24 h. In addition BMP-7 treatment stimulated a 55-fold increase in osteocalcin mRNA as early as 24 h after treatment. A novel finding was that the expression of the chondrocyte markers Sox9 and type II collagen was increased as well. Runx2/Cbfa1 is a molecular switch for osteoblast differentiation. To initiate the study of modulators of Runx2/Cbfa1, such as kinases and cofactors, during osteoblastic differentiation of C2C12 cells treated by BMP-7 in vitro, microarray analyses of gene expressions were performed. Microarray data suggested that a total of 882 transcripts were either up- or downregulated at least 2-fold. Cluster analyses revealed 76 genes (including ESTs) with expression patterns that paralleled Runx2/Cbfa1. Thirteen of these 76 genes were initially selected as potential transcription modulators for further study; including CCAAT/enhancer binding protein delta, distal- less homeobox 1, forkhead box F2, insulin-like growth factor binding protein 4, an ortholog of human osteoclast stimulating factor 1 and p300/CBP-associated factor. Some transcription modulators have been associated with osteoblastic differentiation or interacted with Runx2/Cbfa1. Most of them have not been extensively studied in osteoblastic differentiation and in relationship to Runx2/Cbfa1. Thus, these studies identify potential regulators for Runx2/Cbfa1 and osteoblast differentiation. In addition, our data revealed for the first time that BMP-7 not only induced the expression of osteoblastic differentiation markers but also stimulated the expression of chondroblastic markers in C2C12 cells.

1.
Ahn, M.Y., S.C. Bae, M. Maruyama, Y. Ito (1996) Comparison of the human genomic structure of the Runt domain-encoding PEBP2/CBFalpha gene family. Gene 168: 279–280.
2.
Asahina, I., T.K. Sampath, I. Nishimura, P.V. Hauschka (1993) Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria. J Cell Biol 123: 921–933.
3.
Balint, E., D. Lapointe, H. Drissi, C. Van Der Meijden, D.W. Young, A.J. Van Wijnen, J.L. Stein, G.S. Stein, J.B. Lian (2003) Phenotype discovery by gene expression profiling: Mapping of biological processes linked to BMP-2-mediated osteoblast differentiation. J Cell Biochem 89: 401–426.
4.
Bi, W., J.M. Deng, Z. Zhang, R.R. Behringer, B. de Crombrugghe (1999) Sox9 is required for cartilage formation. Nat Genet 22: 85–89.
5.
Byers, B.A., G.K. Pavlath, T.J. Murphy, G. Karsenty, A.J. Garcia (2002) Cell-type-dependent up-regulation of in vitro mineralization after overexpression of the osteoblast-specific transcription factor Runx2/Cbfal. J Bone Miner Res 17:1931–1944.
6.
Candia, A.F., T. Watabe, S.H. Hawley, D. Onichtchouk, Y. Zhang, R. Derynck, C. Niehrs, K.W. Cho (1997) Cellular interpretation of multiple TGF-beta signals: Intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by Smads. Development 124: 4467–4480.
7.
Chen, X., E. Weisberg, V. Fridmacher, M. Watanabe, G. Naco, M. Whitman (1997) Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389: 85–89.
8.
de Jong, D.S., E.J. van Zoelen, S. Bauerschmidt, W. Olijve, W.T. Steegenga (2002) Microarray analysis of bone morphogenetic protein, transforming growth factor beta, and activin early response genes during osteoblastic cell differentiation. J Bone Miner Res 17: 2119–2129.
9.
Ducy, P. (2000) Cbfa1: A molecular switch in osteoblast biology. Dev Dyn 219: 461–471.
10.
Ducy, P., G. Karsenty (2000) The family of bone morphogenetic proteins. Kidney Int 57: 2207–2214.
11.
Ducy, P., M. Starbuck, M. Priemel, J. Shen, G. Pinero, V. Geoffroy, M. Amling, G. Karsenty (1999) A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 13: 1025–1036.
12.
Ducy, P., R. Zhang, V. Geoffroy, A.L. Ridall, G. Karsenty (1997) Osf2/Cbfa1: A transcriptional activator of osteoblast differentiation. Cell 89: 747–754.
13.
Eisen, M.B., P.T. Spellman, P.O. Brown, D. Botstein (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863–14868.
14.
Enomoto, H., S. Shiojiri, K. Hoshi, T. Furuichi, R. Fukuyama, C.A. Yoshida, N. Kanatani, R. Nakamura, A. Mizuno, A. Zanma, K. Yano, H. Yasuda, K. Higashio, K. Takada, T. Komori (2003) Induction of osteoclast differentiation by Runx2 through RANKL and OPG regulation and partial rescue of osteoclastogenesis in Runx2–/– mice by RANKL transgene. J Biol Chem 278: 23971–23977.
15.
Godin, R.E., E.J. Robertson, A.T. Dudley (1999) Role of BMP family members during kidney development. Int J Dev Biol 43: 405–411.
16.
Gutierrez, S., A. Javed, D.K. Tennant, M. van Rees, M. Montecino, G.S. Stein, J.L. Stein, J.B. Lian (2002) CCAAT/enhancer-binding proteins (C/EBP) beta and delta activate osteocalcin gene transcription and synergize with Runx2 at the C/EBP element to regulate bone-specific expression. J Biol Chem 277: 1316–1323.
17.
Harland, R.M. (1994) The transforming growth factor beta family and induction of the vertebrate mesoderm: Bone morphogenetic proteins are ventral inducers. Proc Natl Acad Sci USA 91: 10243–10246.
18.
Hemmati-Brivanlou, A., D. Melton (1997) Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell 88: 13–17.
19.
Hogan, B.L. (1996) Bone morphogenetic proteins in development. Curr Opin Genet Dev 6: 432–438.
20.
Honda, Y., E.C. Landale, D.D. Strong, D.J. Baylink, S. Mohan (1996) Recombinant synthesis of insulin-like growth factor-binding protein-4 (IGFBP-4): Development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids. J Clin Endocrinol Metab 81: 1389–1396.
21.
Itoh, S., J. Ericsson, J. Nishikawa, C.H. Heldin, P. ten Dijke (2000) The transcriptional co-activator P/CAF potentiates TGF-beta/Smad signaling. Nucleic Acids Res 28: 4291–4298.
22.
Katagiri, T., S. Akiyama, M. Namiki, M. Komaki, A. Yamaguchi, V. Rosen, J.M. Wozney, A. Fujisawa-Sehara, T. Suda (1997) Bone morphogenetic protein-2 inhibits terminal differentiation of myogenic cells by suppressing the transcriptional activity of MyoD and myogenin. Exp Cell Res 230: 342–351.
23.
Katagiri, T., A. Yamaguchi, M. Komaki, E. Abe, N. Takahashi, T. Ikeda, V. Rosen, J.M. Wozney, A. Fujisawa-Sehara, T. Suda (1994) Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol 127: 1755–1766.
24.
Kern, B., J. Shen, M. Starbuck, G. Karsenty (2001) Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J Biol Chem 276: 7101–7107.
25.
Kim, I.S., F. Otto, B. Zabel, S. Mundlos (1999) Regulation of chondrocyte differentiation by Cbfa1. Mech Dev 80: 159–170.
26.
Kingsley, D.M. (1994) The TGF-beta superfamily: New members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8: 133–146.
27.
Knutsen, R., J.E. Wergedal, T.K. Sampath, D.J. Baylink, S. Mohan (1993) Osteogenic protein-1 stimulates proliferation and differentiation of human bone cells in vitro. Biochem Biophys Res Commun 194: 1352–1358.
28.
Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R.T. Bronson, Y.H. Gao, M. Inada, M. Sato, R. Okamoto, Y. Kitamura, S. Yoshiki, T. Kishimoto (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89: 755–764.
29.
Krebsbach, P.H., K. Gu, R.T. Franceschi, R.B. Rutherford (2000) Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Hum Gene Ther 11: 1201–1210.
30.
LaTour, D., S. Mohan, T.A. Linkhart, D.J. Baylink, D.D. Strong (1990) Inhibitory insulin-like growth factor-binding protein: Cloning, complete sequence, and physiological regulation. Mol Endocrinol 4: 1806–1814.
31.
Lechner, M.S., G.R. Dressler (1997) The molecular basis of embryonic kidney development. Mech Dev 62: 105–120.
32.
Levanon, D., V. Negreanu, Y. Bernstein, I. Bar-Am, L. Avivi, Y. Groner (1994) AML1, AML2, and AML3, the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization. Genomics 23: 425–432.
33.
Maas, R., Y.P. Chen, M. Bei, I. Woo, I. Satokata (1996) The role of Msx genes in mammalian development. Ann N Y Acad Sci 785: 171–181.
34.
Maliakal, J.C., I. Asahina, P.V. Hauschka, T.K. Sampath (1994) Osteogenic protein-1 (BMP-7) inhibits cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat osteosarcoma (17/2.8) cells. Growth Factors 11: 227–234.
35.
Manolagas, S.C., D.W. Burton, L.J. Deftos (1981) 1,25-Dihydroxyvitamin D3 stimulates the alkaline phosphatase activity of osteoblast-like cells. J Biol Chem 256: 7115–7117.
36.
Marcotte, E.M., M. Pellegrini, M.J. Thompson, T.O. Yeates, D. Eisenberg (1999) A combined algorithm for genome-wide prediction of protein function. Nature 402: 83–86.
37.
McCarthy, T.L., C. Ji, Y. Chen, K.K. Kim, M. Imagawa, Y. Ito, M. Centrella (2000) Runt domain factor (Runx)-dependent effects on CCAAT/enhancer-binding protein delta expression and activity in osteoblasts. J Biol Chem 275:21746–21753.
38.
Merlo, G.R., B. Zerega, L. Paleari, S. Trombino, S. Mantero, G. Levi (2000) Multiple functions of Dlx genes. Int J Dev Biol 44: 619–626.
39.
Müller, F., H. Rohrer (2002) Molecular control of ciliary neuron development: BMPs and downstream transcriptional control in the parasympathetic lineage. Development 129: 5707–5717.
40.
Mundlos, S., F. Otto, C. Mundlos, J.B. Mulliken, A.S. Aylsworth, S. Albright, D. Lindhout, W.G. Cole, W. Henn, J.H. Knoll, M.J. Owen, R. Mertelsmann, B.U. Zabel, B.R. Olsen (1997) Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89: 773–779.
41.
Ohyama, M., N. Suzuki, Y. Yamaguchi, M. Maeno, K. Otsuka, K. Ito (2002) Effect of enamel matrix derivative on the differentiation of C2C12 cells. J Periodontol 73: 543–550.
42.
Otto, F., A.P. Thornell, T. Crompton, A. Denzel, K.C. Gilmour, I.R. Rosewell, G.W. Stamp, R.S. Beddington, S. Mundlos, B.R. Olsen, P.B. Selby, M.J. Owen (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89: 765–771.
43.
Ozkaynak, E., D.C. Rueger, E.A. Drier, C. Corbett, R.J. Ridge, T.K. Sampath, H. Oppermann (1990) OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO J 9: 2085–2093.
44.
Pouponnot, C., L. Jayaraman, J. Massague (1998) Physical and functional interaction of SMADs and p300/CBP. J Biol Chem 273: 22865–22868.
45.
Reddi, A.H., C.B. Huggins (1975) Formation of bone marrow in fibroblast-transformation ossicles. Proc Natl Acad Sci USA 72: 2212–2216.
46.
Rutherford, R.B., M. Moalli, R.T. Franceschi, D. Wang, K. Gu, P.H. Krebsbach (2002) Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo. Tissue Eng 8: 441–452.
47.
Rutherford, R.B., L. Spangberg, M. Tucker, D. Rueger, M. Charette (1994) The time-course of the induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 39: 833–838.
48.
Rutherford, R.B., J. Wahle, M. Tucker, D. Rueger, M. Charette (1993) Induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 38: 571–576.
49.
Sampath, T.K., J.E. Coughlin, R.M. Whetstone, D. Banach, C. Corbett, R.J. Ridge, E. Ozkaynak, H. Oppermann, D.C. Rueger (1990) Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem 265: 13198–13205.
50.
Sampath, T.K., J.C. Maliakal, P.V. Hauschka, W.K. Jones, H. Sasak, R.F. Tucker, K.H. White, J.E. Coughlin, M.M. Tucker, R.H. Pang et al. (1992) Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem 267: 20352–20362.
51.
Satokata, I., L. Ma, H. Ohshima, M. Bei, I. Woo, K. Nishizawa, T. Maeda, Y. Takano, M. Uchiyama, S. Heaney, H. Peters, Z. Tang, R. Maxson, R. Maas (2000) Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet 24: 391–395.
52.
Satomura, K., P. Krebsbach, P. Bianco, P. Gehron Robey (2000) Osteogenic imprinting upstream of marrow stromal cell differentiation. J Cell Biochem 78: 391–403.
53.
Scharla, S.H., D.D. Strong, C. Rosen, S. Mohan, M. Holick, D.J. Baylink, T.A. Linkhart (1993) 1,25-Dihydroxyvitamin D3 increases secretion of insulin-like growth factor binding protein-4 (IGFBP-4) by human osteoblast-like cells in vitro and elevates IGFBP-4 serum levels in vivo. J Clin Endocrinol Metab 77: 1190–1197.
54.
Selvamurugan, N., W.Y. Chou, A.T. Pearman, M.R. Pulumati, N.C. Partridge (1998) Parathyroid hormone regulates the rat collagenase-3 promoter in osteoblastic cells through the cooperative interaction of the activator protein-1 site and the runt domain binding sequence. J Biol Chem 273: 10647–10657.
55.
Sierra, J., A. Villagra, R. Paredes, F. Cruzat, S. Gutierrez, A. Javed, G. Arriagada, J. Olate, M. Imschenetzky, A.J. Van Wijnen, J.B. Lian, G.S. Stein, J.L. Stein, M. Montecino (2003) Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity. Mol Cell Biol 23: 3339–3351.
56.
Stokes, D.G., G. Liu, R. Dharmavaram, D. Hawkins, S. Piera-Velazquez, S.A. Jimenez (2001) Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Biochem J 360: 461–470.
57.
Theilhaber, J., T. Connolly, S. Roman-Roman, S. Bushnell, A. Jackson, K. Call, T. Garcia, R. Baron (2002) Finding genes in the C2C12 osteogenic pathway by k-nearest-neighbor classification of expression data. Genome Res 12: 165–176.
58.
Thirunavukkarasu, K., D.L. Halladay, R.R. Miles, C.D. Geringer, J.E. Onyia (2002) Analysis of regulator of G-protein signaling-2 (RGS-2) expression and function in osteoblastic cells. J Cell Biochem 85: 837–850.
59.
Tsingotjidou, A., J.M. Nervina, L. Pham, O. Bezouglaia, S. Tetradis (2002) Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3′,5′-monophosphate-mediated pathway in primary neonatal murine osteoblasts. Bone 30: 677–684.
60.
Urist, M.R. (1965) Bone: Formation by autoinduction. Science 150: 893–899.
61.
Urist, M.R., B.S. Strates (1971) Bone morphogenetic protein. J Dent Res 50: 1392–1406.
62.
Vaes, B.L., K.J. Dechering, A. Feijen, J.M. Hendriks, C. Lefevre, C.L. Mummery, W. Olijve, E.J. van Zoelen, W.T. Steegenga (2002) Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development. J Bone Miner Res 17: 2106–2118.
63.
van Gurp, M.F., J. Pratap, M. Luong, A. Javed, H. Hoffmann, A. Giordano, J.L. Stein, E.J. Neufeld, J.B. Lian, G.S. Stein, A.J. van Wijnen (1999) The CCAAT displacement protein/cut homeodomain protein represses osteocalcin gene transcription and forms complexes with the retinoblastoma protein-related protein p107 and cyclin A. Cancer Res 59: 5980–5988.
64.
Wozney, J.M., V. Rosen (1998) Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop: 26–37.
65.
Xiao, G., D. Jiang, R. Gopalakrishnan, R.T. Franceschi (2002) Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J Biol Chem 277: 36181–36187.
66.
Xiao, Z.S., R. Thomas, T.K. Hinson, L.D. Quarles (1998) Genomic structure and isoform expression of the mouse, rat and human Cbfa1/Osf2 transcription factor. Gene 214: 187–197.
67.
Yaffe, D., O. Saxel (1985) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270: 758–766.
68.
Yang, S., D. Wei, D. Wang, M. Phimphilai, P.H. Krebsbach, R.T. Franceschi (2003) In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation. J Bone Miner Res 18: 705–715.
69.
Yeh, L.C., M.L. Adamo, M.S. Olson, J.C. Lee (1997) Osteogenic protein-1 and insulin-like growth factor I synergistically stimulate rat osteoblastic cell differentiation and proliferation. Endocrinology 138: 4181–4190.
70.
Yeh, L.C., A.D. Tsai, J.C. Lee (2002) Osteogenic protein-1 (OP-1, BMP-7) induces osteoblastic cell differentiation of the pluripotent mesenchymal cell line C2C12. J Cell Biochem 87: 292–304.
71.
Zambotti, A., H. Makhluf, J. Shen, P. Ducy (2002) Characterization of an osteoblast-specific enhancer element in the CBFA1 gene. J Biol Chem 277: 41497–41506.
72.
Zhang, M., M. Faugere, H. Malluche, C. Rosen, S. Chernausek, T. Clemens (2003) Paracrine overexpression of igfbp-4 in osteoblasts of transgenic mice decreases bone turnover and causes global growth retardation. J Bone Miner Res 18: 836–843.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.