Molecular and Cellular Biochemistry

, Volume 310, Issue 1–2, pp 241–244 | Cite as

Effect of siRNA targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expression

  • Zhiqing Huang
  • Keying Zhang
  • Xiaoling Chen
  • Jianghong Meng
  • Daiwen Chen


The c-Jun N-terminal kinase (JNK) pathway was reported to be involved in myostatin signaling and MKK4 was suggested as the only upstream kinase for myostatin-induced JNK activation, implying that MKK4 is a suitable target of RNA interference (RNAi) for blocking myostatin activity. The aim of this study was to evaluate the effect of small interfering RNA (siRNA) targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expression. Real-time quantitative PCR revealed that the level of MKK4 expression was efficiently reduced by MKK4-specific siRNA. Western blot assays showed that knockdown of MKK4 attenuated the myostatin-induced downregulation of MyoD and myogenin expression.


Myostatin MKK4 siRNA Differentiation marker gene 



This work was supported by the National Basic Research Program of China under Grant No. 2004CB117506 and was conducted at University of Maryland, College Park, Maryland, USA. We thank Dr. David K.Y. Lei for help in Western blot analysis, and Dr. Jie Zheng for help in cell culture.


  1. 1.
    McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature 387:83–90PubMedCrossRefGoogle Scholar
  2. 2.
    Lin J, Arnold HB, Della-Fera MA, Azain MJ, Hartzell DL, Baile CA (2002) Myostatin knockout in mice increases myogenesis and decreases adipogenesis. Biochem Biophys Res Commun 291:701–706PubMedCrossRefGoogle Scholar
  3. 3.
    Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkinson KN, McPherron AC, Wolfman NM, Lee SJ (2002) Induction of cachexia in mice by systemically administered myostatin. Science 296:1486–1488PubMedCrossRefGoogle Scholar
  4. 4.
    Kambadur R, Sharma M, Smith TP, Bass JJ (1997) Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res 7:910–916PubMedGoogle Scholar
  5. 5.
    McPherron AC, Lee SJ (1997) Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94:12457–12461PubMedCrossRefGoogle Scholar
  6. 6.
    Gonzalez-Cadavid NF, Taylor WE, Yarasheski K, Sinha-Hikim I, Ma K, Ezzat S, Shen R, Lalani R, Asa S, Mamita M, Nair G, Arver S, Bhasin S (1998) Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting. Proc Natl Acad Sci USA 95:14938–14943PubMedCrossRefGoogle Scholar
  7. 7.
    Reardon KA, Davis J, Kapsa RM, Choong P, Byrne E (2001) Myostatin, insulin-like growth factor-1, and leukemia inhibitory factor mRNAs are upregulated in chronic human disuse muscle atrophy. Muscle Nerve 24:893–899PubMedCrossRefGoogle Scholar
  8. 8.
    Lee SJ (2004) Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol 20:61–68PubMedCrossRefGoogle Scholar
  9. 9.
    Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R (2002) Myostatin inhibits myoblast differentiation by downregulating MyoD expression. J Biol Chem 277:49831–49840PubMedCrossRefGoogle Scholar
  10. 10.
    Ríos R, Carneiro I, Arce VM, Devesa J (2002) Myostatin is an inhibitor of myogenic differentiation. Am J Physiol Cell Physiol 282:C993–C999PubMedGoogle Scholar
  11. 11.
    Huang Z, Chen D, Zhang K, Yu B, Chen X, Meng J (2007) Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells. Cell Signal 19:2286–2295PubMedCrossRefGoogle Scholar
  12. 12.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  13. 13.
    Hannon GJ (2002) RNA interference. Nature 418:244–251PubMedCrossRefGoogle Scholar
  14. 14.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498PubMedCrossRefGoogle Scholar
  15. 15.
    Dérijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ (1995) Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267:682–685PubMedCrossRefGoogle Scholar
  16. 16.
    Morighchi T, Toyoshima F, Masuyama N, Hanafusa H, Gotoh Y, Nishida E (1997) A novel SAPK/JNK kinase, MKK7, stimulated by TNFα and cellular stress. EMBO J 16:7045–7053CrossRefGoogle Scholar
  17. 17.
    Tournier C, Whitmarsh AJ, Cavanagh J, Barrett T, Davis RJ (1997) Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc Natl Acad Sci USA 94:7337–7342PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2007

Authors and Affiliations

  • Zhiqing Huang
    • 1
  • Keying Zhang
    • 1
  • Xiaoling Chen
    • 1
  • Jianghong Meng
    • 2
  • Daiwen Chen
    • 1
  1. 1.Institute of Animal NutritionSichuan Agricultural UniversityYaanP.R. China
  2. 2.Department of Nutrition and Food ScienceUniversity of MarylandCollege ParkUSA

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