Skip to main content
SpringerLink
Log in

Overexpression of Six1 gene suppresses proliferation and enhances expression of fast-type muscle genes in C2C12 myoblasts

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Sine oculis homeobox 1 (Six1) homeodomain transcription factor is implicated in the genesis of muscle fiber type diversity, but its regulatory mechanisms on the formation of muscle fiber type are still poorly understood. To elucidate the biological roles of Six1 gene in muscle fiber formation, we established C2C12 cell line overexpressing Six1 and determined the effects of forced Six1 expression on muscle-specific genes expression, cell proliferation, and cell cycles. Our results indicated that Six1 overexpression could significantly promote the expression of fast-type muscle genes Atp2a1, Srl, and Mylpf. Furthermore, Six1 overexpressing C2C12 cells displayed a relative lower proliferative potential, and cell cycle analysis showed that Six1 exerted its role in cell cycle primarily through the regulation of G1/S and G2/M phases. In conclusion, Six1 plays an essential role in modulation of the fast-twitch muscle fiber phenotype through up-regulating fast-type muscle genes expression, and it could suppress the proliferation of muscle cells.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Bassel-Duby RO EN (2006) Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 75:19–37

    Article  Google Scholar 

  2. Chin ER, Olson EN, Richardson JA, Yang Q, Humphries C, Shelton JM, Wu H, Zhu W, Bassel-Duby R, Williams RS (1998) A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev 12:2499–2509

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Chin ER (2005) Role of Ca2+/calmodulin-dependent kinases in skeletal muscle plasticity. J Appl Physiol 99:414–423

    Article  CAS  PubMed  Google Scholar 

  4. Olson EN, Williams RS (2000) Calcineurin signaling and muscle remodeling. Cell 101:689–692

    Article  CAS  PubMed  Google Scholar 

  5. Wu H, Kanatous SB, Thurmond FA, Gallardo T, Isotani E, Bassel-Duby R, Williams RS (2002) Regulation of mitochondrial biogenesis in skeletal muscle by CaMK. Science 296:349–352

    Article  CAS  PubMed  Google Scholar 

  6. Mu XD, Brown LD, Liu YW, Schneider MF (2007) Roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation of cultured adult mouse skeletal muscle fibers. Physiol Genomics 30:300–312

    Article  CAS  PubMed  Google Scholar 

  7. Lin J, Wu H, Tarr PT, Zhang CY, Wu ZD, Boss O, Michael LF, Puigserver P, Isotani E, Olson EN, Lowell BB, Bassel-Duby R, Spiegelman BM (2002) Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature 418:797–801

    Article  CAS  PubMed  Google Scholar 

  8. Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Ham J, Kang H, Evans RM (2004) Regulation of muscle fiber type and running endurance by PPAR delta. PLoS Biol 2:1532–1539

    CAS  Google Scholar 

  9. Schuler M, Ali F, Chambon C, Duteil D, Bornert JM, Tardivel A, Desvergne B, Wahli W, Chambon P, Metzger D (2006) PGC1 alpha expression is controlled in skeletal muscles by PPAR beta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes. Cell Metab 4:407–414

    Article  CAS  PubMed  Google Scholar 

  10. Kim MS, Fielitz J, McAnally J, Shelton JM, Lemon DD, McKinsey TA, Richardson JA, Bassel-Duby R, Olson EN (2008) Protein kinase D1 stimulates MEF2 activity in skeletal muscle and enhances muscle performance. Mol Cell Biol 28:3600–3609

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Olson EN, Spizz G, Tainsky MA (1987) The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation. Mol Cell Biol 7:2104–2111

    CAS  PubMed Central  PubMed  Google Scholar 

  12. Murgia M, Serrano AL, Calabria E, Pallafacchina G, Lømo T, Schiaffino S (2000) Ras is involved in nerve-activity-dependent regulation of muscle genes. Nat Cell Biol 2:142–147

    Article  CAS  PubMed  Google Scholar 

  13. Tsika RW, Schramm C, Simmer G, Fitzsimons DP, Moss RL, Ji J (2008) Overexpression of TEAD-1 in transgenic mouse striated muscles produces a slower skeletal muscle contractile phenotype. J Biol Chem 283:36154–36167

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Liew HP, Choksi SP, Wong KN, Roy S (2008) Specification of vertebrate slow-twitch muscle fiber fate by the transcriptional regulator Blimp1. Dev Biol 324:226–235

    Article  CAS  PubMed  Google Scholar 

  15. Issa LL, Palmer SJ, Guven KL, Santucci N, Hodgson VRM, Popovic K, Joya JE, Hardeman EC (2006) MusTRD can regulate postnatal fiber-specific expression. Dev Biol 293:104–115

    Article  CAS  PubMed  Google Scholar 

  16. Calvo S, Vullhorst D, Venepally P, Cheng J, Karavanova I, Buonanno A (2001) Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription. Mol Cell Biol 21:8490–8503

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Baxendale S, Davison C, Muxworthy C, Wolff C, Ingham PW, Roy S (2004) The B-cell maturation factor Blimp-1 specifies vertebrate slow-twitch muscle fiber identity in response to Hedgehog signaling. Nat Genet 36:88–93

    Article  CAS  PubMed  Google Scholar 

  18. von Hofsten J, Elworthy S, Gilchrist MJ, Smith JC, Wardle FC, Ingham PW (2008) Prdm1- and Sox6-mediated transcriptional repression specifies muscle fibre type in the zebrafish embryo. EMBO Rep 9:683–689

    Article  Google Scholar 

  19. Polly P, Haddadi LM, Issa LL, Subramaniam N, Palmer SJ, Tay ESE, Hardeman EC (2003) hMusTRD1 alpha 1 represses MEF2 activation of the troponin I slow enhancer. J Biol Chem 278:36603–36610

    Article  CAS  PubMed  Google Scholar 

  20. Hagiwara N, Yeh M, Liu A (2007) Sox6 is required for normal fiber type differentiation of fetal skeletal muscle in mice. Dev Dyn 236:2062–2076

    Article  CAS  PubMed  Google Scholar 

  21. Hamade A, Deries M, Begemann G, Bally-Cuif L, Genet C, Sabatier F, Bonnieu A, Cousin X (2006) Retinoic acid activates myogenesis in vivo through Fgf8 signalling. Dev Biol 289:127–140

    Article  CAS  PubMed  Google Scholar 

  22. Groves JA, Hammond CL, Hughes SM (2005) Fgf8 drives myogenic progression of a novel lateral fast muscle fibre population in zebrafish. Development 132:4211–4222

    Article  CAS  PubMed  Google Scholar 

  23. Maves L, Waskiewicz AJ, Paul B, Cao Y, Tyler A, Moens CB, Tapscott SJ (2007) Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation. Development 134:3371–3382

    Article  CAS  PubMed  Google Scholar 

  24. Arany Z, Lebrasseur N, Morris C, Smith E, Yang WL, Ma YH, Chin S, Spiegelman BM (2007) The transcriptional coactivator PGC-1 beta drives the formation of oxidative type IIX fibers in skeletal muscle. Cell Metab 5:35–46

    Article  CAS  PubMed  Google Scholar 

  25. Shi H, Scheffler JM, Pleitner JM, Zeng C, Park S, Hannon KM, Grant AL, Gerrard DE (2008) Modulation of skeletal muscle fiber type by mitogen-activated protein kinase signaling. FASEB J 22:2990–3000

    Article  CAS  PubMed  Google Scholar 

  26. Laclef C, Hamard G, Demignon J, Souil E, Houbron C, Maire P (2003) Altered myogenesis in Six1-deficient mice. Development 130:2239–2252

    Article  CAS  PubMed  Google Scholar 

  27. Grifone R, Laclef C, Spitz F, Lopez S, Demignon J, Guidotti JE, Kawakami K, Xu PX, Kelly R, Petrof BJ, Daegelen D, Concordet JP, Maire P (2004) Six1 and Eya1 expression can reprogram adult muscle from the slow-twitch phenotype into the fast-twitch phenotype. Mol Cell Biol 24:6253–6267

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Bessarab DA, Chong SW, Srinivas BP, Korzh V (2008) Six1a is required for the onset of fast muscle differentiation in zebrafish. Dev Biol 323:216–228

    Article  CAS  PubMed  Google Scholar 

  29. Niro C, Demignon J, Vincent S, Liu YB, Giordani J, Sgarioto N, Favier M, Guillet-Deniau I, Blais A, Maire P (2010) Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome. Dev Biol 338:168–182

    Article  CAS  PubMed  Google Scholar 

  30. Richard AF, Demignon J, Sakakibara I, Pujol J, Favier M, Strochlic L, Le Grand F, Sgarioto N, Guernec A, Schmitt A, Cagnard N, Huang R, Legay C, Guillet-Deniau I, Maire P (2011) Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression. Dev Biol 359:303–320

    Article  CAS  PubMed  Google Scholar 

  31. Wu W, Ren Z, Wang Y, Chao Z, Xu D, Xiong Y (2011) Molecular characterization, expression patterns and polymorphism analysis of porcine Six1 gene. Mol Biol Rep 38:2619–2632

    Article  CAS  PubMed  Google Scholar 

  32. Himeda CL, Ranish JA, Angello JC, Maire P, Aebersold R, Hauschka SD (2004) Quantitative proteomic identification of Six4 as the trex-binding factor in the muscle creatine kinase enhancer. Mol Cell Biol 24:2132–2143

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Salminen M, Lopez S, Maire P, Kahn A, Daegelen D (1996) Fast-muscle-specific DNA-protein interactions occurring in vivo at the human aldolase A M promoter are necessary for correct promoter activity in transgenic mice. Mol Cell Biol 16:76–85

    CAS  PubMed Central  PubMed  Google Scholar 

  34. Spitz F, Salminen M, Demignon J, Kahn A, Daegelen D, Maire P (1997) A combination of MEF3 and NFI proteins activates transcription in a subset of fast-twitch muscles. Mol Cell Biol 17:656–666

    CAS  PubMed Central  PubMed  Google Scholar 

  35. Grifone R, Demignon J, Houbron C, Souil E, Niro C, Seller MJ, Hamard G, Maire P (2005) Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo. Development 132:2235–2249

    Article  CAS  PubMed  Google Scholar 

  36. Ridgeway AG, Skerjanc IS (2001) Pax3 is essential for skeletal myogenesis and the expression of Six1 and Eya2. J Biol Chem 276:19033–19039

    Article  CAS  PubMed  Google Scholar 

  37. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  38. Giordani J, Bajard L, Demignon J, Daubas P, Buckingham M, Maire P (2007) Six proteins regulate the activation of Myf5 expression in embryonic mouse limbs. Proc Natl Acad Sci USA 104:11310–11315

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Spitz F, Demignon J, Porteu A, Kahn A, Concordet JP, Daegelen D, Maire P (1998) Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site. Proc Natl Acad Sci USA 95:14220–14225

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Ott MO, Bober E, Lyons G, Arnold H, Buckingham M (1991) Early expression of the myogenic regulatory gene, Myf-5, in precursor cells of skeletal muscle in the mouse embryo. Development 111:1097–1107

    CAS  PubMed  Google Scholar 

  41. Rudnicki MA, Braun T, Hinuma S, Jaenisch R (1992) Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell 71:383–390

    Article  CAS  PubMed  Google Scholar 

  42. Sassoon D, Lyons G, Wright WE, Lin V, Lassar A, Weintraub H, Buckingham M (1989) Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 341:303–307

    Article  CAS  PubMed  Google Scholar 

  43. Bober E, Lyons GE, Braun T, Cossu G, Buckingham M, Arnold HH (1991) The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development. J Cell Biol 113:1255–1265

    Article  CAS  PubMed  Google Scholar 

  44. Kawakami K, Sato S, Ozaki H, Ikeda K (2000) Six family genes-structure and function as transcription factors and their roles in development. Bioessays 22:616–626

    Article  CAS  PubMed  Google Scholar 

  45. Kucharczuk KL, Love CM, Dougherty NM, Goldhamer DJ (1999) Fine-scale transgenic mapping of the MyoD core enhancer: MyoD is regulated by distinct but overlapping mechanisms in myotomal and non-myotomal muscle lineages. Development 126:1957–1965

    CAS  PubMed  Google Scholar 

  46. Maire P, Gautron S, Hakim V, Gregori C, Mennecier F, Kahn A (1987) Characterization of three optional promoters in the 5′ region of the human aldolase A gene. J Mol Biol 197:425–438

    Article  CAS  PubMed  Google Scholar 

  47. Behbakht K, Qamar L, Aldridge CS, Coletta RD, Davidson SA, Thorburn A, Ford HL (2007) Six1 overexpression in ovarian carcinoma causes resistance to TRAIL-mediated apoptosis and is associated with poor survival. Cancer Res 67:3036–3042

    Article  CAS  PubMed  Google Scholar 

  48. Coletta RD, Christensen K, Reichenberger KJ, Lamb J, Micomonaco D, Huang LL, Wolf DM, Muller-Tidow C, Golub TR, Kawakami K, Ford HL (2004) The Six1 homeoprotein stimulates tumorigenesis by reactivation of cyclin A1. Proc Natl Acad Sci USA 101:6478–6483

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Ford HL, Kabingu EN, Bump EA, Mutter GL, Pardee AB (1998) Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis. Proc Natl Acad Sci USA 95:12608–12613

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Reichenberger KJ, Coletta RD, Schulte AP, Varella-Garcia M, Ford HL (2005) Gene amplification is a mechanism of Six1 overexpression in breast cancer. Cancer Res 65:2668–2675

    Article  CAS  PubMed  Google Scholar 

  51. Yu YL, Khan J, Khanna C, Helman L, Meltzer PS, Merlino G (2004) Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 10:175–181

    Article  CAS  PubMed  Google Scholar 

  52. Khan J, Bittner ML, Saal LH, Teichmann U, Azorsa DO, Gooden GC, Pavan WJ, Trent JM, Meltzer PS (1999) cDNA microarrays detect activation of a myogenic transcription program by the PAX3–FKHR fusion oncogene. Proc Natl Acad Sci USA 96:13264–13269

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Ford HL, Landesman-Bollag E, Dacwag CS, Stukenberg PT, Pardee AB, Seldin DC (2000) Cell cycle-regulated phosphorylation of the human SIX1 homeodomain protein. J Biol Chem 275:22245–22254

    Article  CAS  PubMed  Google Scholar 

  54. Goranov AI, Cook M, Ricicova M, Ben-Ari G, Gonzalez C, Hansen C, Tyers M, Amon A (2009) The rate of cell growth is governed by cell cycle stage. Genes Dev 23:1408–1422

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This research program was funded by the Youth Science and Technology Innovation Fund of Nanjing Agricultural College (KJ2012014), National Natural Science Foundation of China (31000996), and the National Project for Breeding of Transgenic Pig (2008ZX08006-002).

Author information

Authors and Affiliations

  1. Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China

    Wangjun Wu

  2. Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science, Huazhong Agricultural University, Wuhan, 430070, Hubei, China

    Wangjun Wu, Zhuqing Ren, Lin Zhang, Yang Liu & Yuanzhu Xiong

  3. Qingdao Institute of Animal Science and Veterinary Medicine, Qingdao, 266100, Shandong, China

    Hegang Li

Authors
  1. Wangjun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhuqing Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hegang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuanzhu Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wangjun Wu or Yuanzhu Xiong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, W., Ren, Z., Zhang, L. et al. Overexpression of Six1 gene suppresses proliferation and enhances expression of fast-type muscle genes in C2C12 myoblasts. Mol Cell Biochem 380, 23–32 (2013). https://doi.org/10.1007/s11010-013-1653-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-013-1653-3

Keywords

Search

Navigation