Skip to main content

Advertisement

SpringerLink
Log in

δEF1 represses BMP-2-induced differentiation of C2C12 myoblasts into the osteoblast lineage

  • Published:
Journal of Biomedical Science

Abstract

Osteoblasts, derived from pluripotent mesenchymal precursor cells, acquire their differentiated phenotypes under the control of a series of regulatory factors, the best known of which is BMP-2. Our recent preliminary studies suggest that expression of δEF1, a member of the zinc finger-homeodomain transcription factor family, is significantly down-regulated as human mesenchymal stem cells (MSCs) are subjected to osteoblastic differentiation in the presence of BMP-2. Here we demonstrate that overexpression of δEF1 in murine pre-myoblast C2C12 cells resulted in a decrease in the mRNA levels of early osteoblast marker genes induced by BMP-2 including osterix and collagen type I. This inhibitory effect was further confirmed by decreased alkaline phosphatase (ALP) activities. Neither of the zinc finger clusters of δEF1 is necessary for its repressive effect on BMP-2-induced osteoblastic differentiation of C2C12 cells. Immunoprecipitation results indicated that δEF1 did not physically associate with Smads proteins, suggesting that the inhibitory effect of δEF1 may be Smad-independent. δEF1 overexpression in C2C12 cells resulted in down-regulation of activating protein-1 (AP-1) activities promoted by BMP-2. Moreover, δEF1 exhibited transrepression on murine osteocalcin gene which effect is partially mediated through diminishing of AP-1 signaling. These results suggest that δEF1 acts as a potent inhibitor of BMP-2-induced osteogenesis in vitro, in part, by differentially regulating the AP-1 signaling pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ten Dijke P., Miyazono K., Heldin C.H. (2000) Signaling inputs converge on nuclear effectors in TGF-beta signaling. Trends Biochem. Sci. 25: 64–70

    Article  PubMed  CAS  Google Scholar 

  2. Wozney J.M., Rosen V. (1998) Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin. Orthop. Relat. Res. 346: 26–37

    Article  PubMed  Google Scholar 

  3. Franceschi R.T., Xiao G. (2003) Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J. Cell. Biochem. 88: 446–454

    Article  PubMed  CAS  Google Scholar 

  4. Kim Y.J., Lee M.H., Wozney J.M., Cho J.Y., Ryoo H.M. (2004) Bone morphogenetic protein-2-induced alkaline phosphatase expression is stimulated by Dlx5 and repressed by Msx2. J. Biol. Chem. 279: 50773–50780

    Article  PubMed  CAS  Google Scholar 

  5. Nakashima K., Zhou X., Kunkel G., Zhang Z., Deng J.M., Behringer R.R., de Crombrugghe B. (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108: 17–29

    Article  PubMed  CAS  Google Scholar 

  6. Olney R.C. (2003) Regulation of bone mass by growth hormone. Med.. Pediatr. Oncol. 41: 228–234

    Article  PubMed  Google Scholar 

  7. Katagiri T., Yamaguchi A., Komaki M., Abe E., Takahashi N., Ikeda T., Rosen V., Wozney J.M., Fujisawa-Sehara A., Suda T. (1994) Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127: 1755–1766

    Article  PubMed  CAS  Google Scholar 

  8. Massague J., Wotton D. (2000) Transcriptional control by the TGF-beta/Smad signaling system. EMBO J. 19: 1745–1754

    Article  PubMed  CAS  Google Scholar 

  9. Wrana J.L., Attisano L. (2000) The Smad pathway. Cytokine Growth Factor Rev. 11: 5–13

    Article  PubMed  CAS  Google Scholar 

  10. Miyazono K., Kusanagi K., Inoue H. (2001) Divergence and convergence of TGF-β/BMP signaling. J. Cell. Physiol. 187: 265–276

    Article  PubMed  CAS  Google Scholar 

  11. Attisano L., Wrana J.L. (2002) Signal transduction by the TGF-ß superfamily. Science 296: 1646–1647

    Article  PubMed  CAS  Google Scholar 

  12. Lai C.F., Cheng S.L. (2002) Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells. J. Biol. Chem. 277: 15514–15522

    Article  PubMed  CAS  Google Scholar 

  13. Celil A.B., Campbell P.G. (2005) BMP-2 and insulin-like growth factor-I mediate osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J. Biol. Chem. 280: 31353–31359

    Article  PubMed  CAS  Google Scholar 

  14. Naito J., Kaji H., Sowa H., Hendy G.N., Sugimoto T., Chihara K. (2005) Menin Suppresses Osteoblast Differentiation by Antagonizing the AP-1 Factor, JunD. J. Biol. Chem. 280: 4785–4791

    Article  PubMed  CAS  Google Scholar 

  15. Palcy S., Goltzman D. (1999) Protein kinase signalling pathways involved in the up-regulation of the rat alpha1(I) collagen gene by transforming growth factor beta1 and bone morphogenetic protein 2 in osteoblastic cells. Biochem. J. 343: 21–27

    Article  PubMed  CAS  Google Scholar 

  16. Gallea S., Lallemand F., Atfi A., Rawadi G., Ramez V., Spinella-Jaegle S., Kawai S., Faucheu C., Huet L., Baron R., Roman-Roman S. (2001) Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone 28: 491–498

    Article  PubMed  CAS  Google Scholar 

  17. Lee K.S., Hong S.H., Bae S.C. (2002) Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene. 21: 7156–7163

    Article  PubMed  CAS  Google Scholar 

  18. Celil A.B., Hollinger J.O., Campbell P.G. (2005) Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. J. Cell. Biolchem. 95: 518–528

    Article  CAS  Google Scholar 

  19. Fortini M.E., Lai Z.C., Rubin G.M. (1991) The Drosophila zfh-1 and zfh-2 genes encode novel proteins containing both zinc-finger and homeodomain motifs. Mech. Dev. 34: 113–122

    Article  PubMed  CAS  Google Scholar 

  20. Lai Z.C., Rushton E., Bate M., Rubin G.M. (1993) Loss of function of the Drosophila zfh-1 gene results in abnormal development of mesodermally derived tissues. Proc. Natl. Acad. Sci. U.S.A. 90: 4122–4126

    Article  PubMed  CAS  Google Scholar 

  21. Verschueren K., Remacle J.E., Collart C., Kraft H., Baker B.S., Tylzanowski P., Nelles L., Wuytens G., Su M.T., Bodmer R., Smith J.C., Huylebroeck D. (1999) SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5′-CACCT sequences in candidate target genes. J. Biol. Chem. 274: 20489–20498

    Article  PubMed  CAS  Google Scholar 

  22. Watanabe Y., Kawakami K., Hirayama Y., Nagano K. (1993) Transcription factors positively and negatively regulating the Na,K-ATPase alpha 1 subunit gene. J. Biochem. 114: 849–855

    PubMed  CAS  Google Scholar 

  23. Funahashi J., Kamachi Y., Goto K., Kondoh H. (1991) Identification of nuclear factor delta EF1 and its binding site essential for lens-specific activity of the delta 1-crystallin enhancer. Nucleic Acids Res. 19: 3543–3547

    Article  PubMed  CAS  Google Scholar 

  24. Funahashi J., Sekido R., Murai K., Kamachi Y., Kondoh H. (1993) Delta-crystallin enhancer binding protein delta EF1 is a zinc finger-homeodomain protein implicated in postgastrulation embryogenesis. Development 119: 433–446

    PubMed  CAS  Google Scholar 

  25. Takagi T., Moribe H., Kondoh H., Higashi Y. (1998) DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages. Development 125: 21–31

    PubMed  CAS  Google Scholar 

  26. Sekido R., Takagi T., Okanami M., Moribe H., Yamamura M., Higashi Y., Kondoh H. (1996) Organization of the gene encoding transcriptional repressor deltaEF1 and cross-species conservation of its domains. Gene 173: 227–232

    Article  PubMed  CAS  Google Scholar 

  27. Franklin A.J., Jetton T.L., Shelton K.D., Magnuson M.A. (1994) BZP, a novel serum-responsive zinc finger protein that inhibits gene transcription. Mol. Cell. Biol. 14: 6773–6788

    PubMed  CAS  Google Scholar 

  28. Kamachi Y., Kondoh H. (1993) Overlapping positive and negative regulatory elements determine lens-specific activity of the delta 1-crystallin enhancer. Mol. Cell. Biol. 13: 5206–5215

    PubMed  CAS  Google Scholar 

  29. Sekido R., Murai K., Kamachi Y., Kondoh H. (1997) Two mechanisms in the action of repressor deltaEF1: binding site competition with an activator and active repression. Genes Cells 2: 771–783

    Article  PubMed  CAS  Google Scholar 

  30. Sekido R., Murai K., Funahashi J., Kamachi Y., Fujisawa-Sehara A., Nabeshima Y., Kondoh H. (1994) The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation. Mol. Cell. Biol. 14: 5692–5700

    PubMed  CAS  Google Scholar 

  31. Postigo A.A., Dean D.C. (1997) ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation. EMBO J. 16: 3935–3943

    Article  PubMed  CAS  Google Scholar 

  32. Eger A., Aigner K., Sonderegger S., Dampier B., Oehler S., Schreiber M., Berx G., Cano A., Beug H., Foisner R. (2005) DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 24: 2375–2385

    Article  PubMed  CAS  Google Scholar 

  33. Higashi Y., Moribe H., Takagi T., Sekido R., Kawakami K., Kikutani H., Kondoh H. (1997) Impairment of T cell development in deltaEF1 mutant mice. J. Exp. Med. 185: 1467–1479

    Article  PubMed  CAS  Google Scholar 

  34. Terraz C., Toman D., Delauche M., Ronco P., Rossert J. (2001) delta Ef1 binds to a far upstream sequence of the mouse pro-alpha 1(I) collagen gene and represses its expression in osteoblasts. J. Biol. Chem. 276: 37011–37019

    Article  PubMed  CAS  Google Scholar 

  35. Murray D., Precht P., Balakir R., Horton WE Jr. (2000) The transcription factor deltaEF1 is inversely expressed with type II collagen mRNA and can repress Col2a1 promoter activity in transfected chondrocytes. J. Biol. Chem. 275: 3610–3618

    Article  PubMed  CAS  Google Scholar 

  36. Sooy K., Demay M.B. (2002) Transcriptional repression of the rat osteocalcin gene by deltaEF1. Endocrinology 143: 3370–3375

    Article  PubMed  CAS  Google Scholar 

  37. Furusawa T., Moribe H., Kondoh H. and Higashi Y., Identification of CtBP1 and CtBP2 as corepressors of zinc finger-homeodomain factor deltaEF1. Mol. Cell. Biol. 19: 8581–8590, 1999

    Google Scholar 

  38. Chamberlain EM, Sanders MM (1999) Identification of the novel player δEF1 in estrogen transcriptional cascades. Mol. Cell. Biol. 19: 3600–3606

    PubMed  CAS  Google Scholar 

  39. Lazarova D.L., Bordonaro M., Sartorelli A.C. (2001) Transcriptional regulation of the vitamin D(3) receptor gene by ZEB. Cell Growth Differ. 12: 319–326

    PubMed  CAS  Google Scholar 

  40. Prashar Y., Weissman S.M. (1996) Analysis of differential gene expression by display of 3′ end restriction fragments of cDNAs. Proc. Natl. Acad. Sci. U.S.A. 93: 659–663

    Article  PubMed  CAS  Google Scholar 

  41. Lee M.H., Javed A., Kim H.J., Shin H.I., Gutierrez S., Choi J.Y., Rosen V., Stein J.L., Wijnen A.J., Stein G.S., Lian J.B., Ryoo H.M. (1999) Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation. J. Cell. Biochem. 73: 114–125

    Article  PubMed  CAS  Google Scholar 

  42. Lee K.S., Kim H.J., Li Q.L., Chi X.Z., Ueta C., Komori T., Wozney J.M., Kim E.G., Cho J.Y., Ryoo H.M., Bae S.C. (2000) Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol. Cell. Biol. 20: 8783–8792

    Article  PubMed  CAS  Google Scholar 

  43. Nakashima K., Zhou X., Kunkel G., Zhang Z., Deng J.M., Behringer R.R., de Crombrugghe B. (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108: 17–29

    Article  PubMed  CAS  Google Scholar 

  44. Shi Y., Massague J. (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113: 685–700

    Article  PubMed  CAS  Google Scholar 

  45. Bai X.C., Lu D., Bai J., Zheng H., Ke Z.Y., Li X.M., Luo S.Q. (2004) Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-κB. Biochem. Biophys. Res. Commun. 314: 197–207

    Article  PubMed  CAS  Google Scholar 

  46. Lu X., Gilbert L., He X., Rubin J., Nanes M.S. (2006) Transcriptional regulation of the osterix (Osx, Sp7) promoter by tumor necrosis factor identifies disparate effects of mitogen-activated protein kinase and NFκB pathways. J. Biol. Chem. 281: 6297–6306

    Article  PubMed  CAS  Google Scholar 

  47. Reddi A.H. (1998) Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat. Biotechnol. 16: 247–252

    Article  PubMed  CAS  Google Scholar 

  48. Postigo A.A. (2003) Opposing functions of ZEB proteins in the regulation of the TGFbeta/BMP signaling pathway. EMBO J. 22: 2443–2452

    Article  PubMed  CAS  Google Scholar 

  49. Postigo A.A., Depp J.L., Taylor J.J., Kroll K.L. (2003) Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. EMBO J. 22: 2453–2462

    Article  PubMed  CAS  Google Scholar 

  50. Turner J., Crossley M. (2001) The CtBP family: enigmatic and enzymatic transcriptional co-repressors. Bioessays. 23: 683–690

    Article  PubMed  CAS  Google Scholar 

  51. Chinnadurai G. (2002) CtBP, an unconventional transcriptional corepressor in development and oncogenesis. Mol. Cell 9: 213–224

    Article  PubMed  CAS  Google Scholar 

  52. Ikeda K., Halle J.P., Stelzer G., Meisterernst M., Kawakami K. (1998) Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6. Mol. Cell. Biol. 18: 10–18

    PubMed  CAS  Google Scholar 

  53. Ayse B.C., Jeffrey O.H., Phil G.C. (2005) Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. J. Cell Biol. 95: 518–528

    Google Scholar 

  54. Jochum W., David J.P., Elliott C., Wutz A., Plenk H. Jr., Matsuo K., Wagner E.F. (2000) Increased bone formation and osteosclerosis in mice overexpressing the transcription factor Fra-1. Nat. Med. 6: 980–984

    Article  PubMed  CAS  Google Scholar 

  55. Sabatakos G., Sims N.A., Chen J., Aoki K., Kelz M., Amling M., Bouali Y., Mukhopadhyay K., Ford K., Nestler E., Baron R. (2000) Overexpression of DeltaFosB transcription factor(s) increases bone formation and inhibits adipogenesis. Nat. Med. 6: 985–990

    Article  PubMed  CAS  Google Scholar 

  56. Iotsova V., Caamano J., Loy J., Yang Y., Lewin A., Bravo R. (1997) Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat. Med.. 3: 1285–1289

    Article  PubMed  CAS  Google Scholar 

  57. Nanes M.S., Kuno H., Demay M.B., Kurian M., Hendy G.N., DeLuca H.F., Titus L., Rubin J. (1994) A single up-stream element confers responsiveness to 1,25-dihydroxy- vitamin D3 and tumor necrosis factor-alpha in the rat osteocalcin gene. Endocrinology 134: 1113–1120

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. G. Sabatakos, Skeletal and Inflammatory Diseases, Procter and Gamble Pharmaceuticals, USA, for the project assistance and critical reading on the manuscript and providing Smads and ALK-6 expression plasmids. We thank M. Petrey and P. Stevens, Skeletal and Inflammatory Diseases, Procter and Gamble Pharmaceuticals, USA, for their technical help. We thank Dr. D. Chen, Center for Musculoskeletal Research, University of Rochester Medical Center, USA, for providing osteocalcin reporter and Runx2 expression plasmid. This work is supported by a grant from The National Nature Science Foundation of China (No. 30640029).

Author information

Authors and Affiliations

  1. Medical College of Nankai University, 94 Weijin Road, Tianjin, 300071, China

    Shuang Yang, Li Zhao, Juhua Yang, Dinggeng Chai, Ming Zhang, Jie Zhang & Tianhui Zhu

  2. Skeletal and Inflammatory Diseases, Procter and Gamble Pharmaceuticals, Mason, USA

    Xiaohui Ji

Authors
  1. Shuang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Li Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juhua Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dinggeng Chai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaohui Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tianhui Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tianhui Zhu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, S., Zhao, L., Yang, J. et al. δEF1 represses BMP-2-induced differentiation of C2C12 myoblasts into the osteoblast lineage. J Biomed Sci 14, 663–679 (2007). https://doi.org/10.1007/s11373-007-9155-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11373-007-9155-5

Keywords

Search

Navigation