Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis

Abstract

Premature fusion of one or more of the cranial sutures (craniosynostosis) in humans causes over 100 skeletal diseases, which occur in 1 of 2,500 live births1,2,3. Among them is Apert syndrome, one of the most severe forms of craniosynostosis, primarily caused by missense mutations leading to amino acid changes S252W or P253R in fibroblast growth factor receptor 2 (FGFR2)4,5,6. Here we show that a small hairpin RNA targeting the dominant mutant form of Fgfr2 (Fgfr2S252W) completely prevents Apert-like syndrome in mice. Restoration of normal FGFR2 signaling is manifested by an alteration of the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2), implicating the gene encoding ERK and the genes downstream of it in disease expressivity. Furthermore, treatment of the mutant mice with U0126, an inhibitor of mitogen-activated protein (MAP) kinase kinase 1 and 2 (MEK1/2) that blocks phosphorylation and activation of ERK1/2, significantly inhibits craniosynostosis. These results illustrate a pathogenic role for ERK activation in craniosynostosis resulting from FGFR2 with the S252W substitution and introduce a new concept of small-molecule inhibitor–mediated prevention and therapy for diseases caused by gain-of-function mutations in the human genome.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Rescue of Apert syndrome–like craniosynostosis by shRNA.
Figure 2: Evaluation of changes in mice treated with shRNA and dynamics of FGFR2 signaling.
Figure 3: Treatment with U0126 repressed Apert syndrome–like craniosynostosis in mice.
Figure 4: ERK phosphorylation and gene expression of wild-type and R2-S252W mutant mice upon treatment with U0126.
Figure 5: Repression of the craniosynostosis phenotype of R2-S252W mutant mice with U0126.

References

  1. Katzen, J.T. & McCarthy, J.G. Syndromes involving craniosynostosis and midface hypoplasia. Otolaryngol Clin. North Am. 33, 1257–1284 (2000).

    Article  CAS  Google Scholar 

  2. Muenke, M. & Schell, U. Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet. 11, 308–313 (1995).

    Article  CAS  Google Scholar 

  3. Renier, D., Lajeunie, E., Arnaud, E. & Marchac, D. Management of craniosynostoses. Childs Nerv. Syst. 16, 645–658 (2000).

    Article  CAS  Google Scholar 

  4. Ibrahimi, O.A., Chiu, E.S., McCarthy, J.G. & Mohammadi, M. Understanding the molecular basis of Apert syndrome. Plast. Reconstr. Surg. 115, 264–270 (2005).

    CAS  PubMed  Google Scholar 

  5. Moloney, D.M. et al. Exclusive paternal origin of new mutations in Apert syndrome. Nat. Genet. 13, 48–53 (1996).

    Article  CAS  Google Scholar 

  6. Park, W.J. et al. Analysis of phenotypic features and FGFR2 mutations in Apert syndrome. Am. J. Hum. Genet. 57, 321–328 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Coumoul, X. & Deng, C.X. Roles of FGF receptors in mammalian development and congenital diseases. Birth Defects Res. C Embryo Today 69, 286–304 (2003).

    Article  CAS  Google Scholar 

  8. Ornitz, D.M. & Itoh, N. Fibroblast growth factors. Genome Biol. 2 REVIEWS3005 (2001) (doi:10.1186/gb-2001-2-3-reviews3005).

    Google Scholar 

  9. Powers, C.J., McLeskey, S.W. & Wellstein, A. Fibroblast growth factors, their receptors and signaling. Endocr. Relat. Cancer 7, 165–197 (2000).

    Article  CAS  Google Scholar 

  10. Chen, L. & Deng, C.X. Roles of FGF signaling in skeletal development and human genetic diseases. Front. Biosci. 10, 1961–1976 (2005).

    Article  CAS  Google Scholar 

  11. McIntosh, I., Bellus, G.A. & Jab, E.W. The pleiotropic effects of fibroblast growth factor receptors in mammalian development. Cell Struct. Funct. 25, 85–96 (2000).

    Article  CAS  Google Scholar 

  12. Chen, L., Li, D., Li, C., Engel, A. & Deng, C.X.A. Ser252Trp substitution in mouse fibroblast growth factor receptor 2 (Fgfr2) results in craniosynostosis. Bone 33, 169–178 (2003).

    Article  CAS  Google Scholar 

  13. Lakso, M. et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865 (1996).

    Article  CAS  Google Scholar 

  14. Coumoul, X., Shukla, V., Li, C., Wang, R.H. & Deng, C.X. Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference. Nucleic Acids Res. 33, e102 (2005) (doi:10.1093/nar/gni100).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Coumoul, X., Li, W., Wang, R.H. & Deng, C. Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system. Nucleic Acids Res. 32, e85 (2004).

    Article  Google Scholar 

  16. Ralph, G.S. et al. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat. Med. 11, 429–433 (2005).

    Article  CAS  Google Scholar 

  17. Chikazu, D. et al. Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase. J. Biol. Chem. 275, 31444–31450 (2000).

    Article  CAS  Google Scholar 

  18. Xiao, G., Jiang, D., Gopalakrishnan, R. & Franceschi, R.T. 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 (2002).

    Article  CAS  Google Scholar 

  19. Chaudhary, L.R. & Avioli, L.V. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) by FGF-2 and PDGF-BB in normal human osteoblastic and bone marrow stromal cells: differences in mobility and in-gel renaturation of ERK1 in human, rat, and mouse osteoblastic cells. Biochem. Biophys. Res. Commun. 238, 134–139 (1997).

    Article  CAS  Google Scholar 

  20. Spector, J.A. et al. FGF-2 acts through an ERK1/2 intracellular pathway to affect osteoblast differentiation. Plast. Reconstr. Surg. 115, 838–852 (2005).

    Article  CAS  Google Scholar 

  21. Kim, H.J. et al. Erk pathway and activator protein 1 play crucial roles in FGF2-stimulated premature cranial suture closure. Dev. Dyn. 227, 335–346 (2003).

    Article  CAS  Google Scholar 

  22. Rubinfeld, H. & Seger, R. The ERK cascade: a prototype of MAPK signaling. Mol. Biotechnol. 31, 151–174 (2005).

    Article  CAS  Google Scholar 

  23. Cabrita, M.A. & Christofori, G. Sprouty proteins: antagonists of endothelial cell signaling and more. Thromb. Haemost. 90, 586–590 (2003).

    Article  CAS  Google Scholar 

  24. Smith, T.G., Sweetman, D., Patterson, M., Keyse, S.M. & Munsterberg, A. Feedback interactions between MKP3 and ERK MAP kinase control scleraxis expression and the specification of rib progenitors in the developing chick somite. Development 132, 1305–1314 (2005).

    Article  CAS  Google Scholar 

  25. Furthauer, M., Lin, W., Ang, S.L., Thisse, B. & Thisse, C. Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat. Cell Biol. 4, 170–174 (2002).

    Article  CAS  Google Scholar 

  26. Favata, M.F. et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623–18632 (1998).

    Article  CAS  Google Scholar 

  27. Perlyn, C.A., Morriss-Kay, G., Darvann, T., Tenenbaum, M. & Ornitz, D.M. A model for the pharmacological treatment of Crouzon syndrome. Neurosurgery 59, 210–215 (2006).

    Article  Google Scholar 

  28. Eswarakumar, V.P. et al. Attenuation of signaling pathways stimulated by pathologically activated FGF-receptor 2 mutants prevents craniosynostosis. Proc. Natl. Acad. Sci. USA 103, 18603–18608 (2006).

    Article  CAS  Google Scholar 

  29. Sui, G. et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–5520 (2002).

    Article  CAS  Google Scholar 

  30. Yang, X. et al. TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage. J. Cell Biol. 153, 35–46 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank X. Xu, C. Li and L. Cao for critical discussions. We thank T. Fishler, J. De Soto, J. Miller and A. McPherron for critically reading the manuscript. This work was supported by the Intramural Research Program of the National Institute of Diabetes, Digestive and Kidney Diseases (US National Institutes of Health).

Author information

Authors and Affiliations

Authors

Contributions

V.S. performed most experiments, X.C. generated the U6-Fgfr2S252W shRNA transgenic mice and performed shRNA-related experiments, R.-H.W. and H.-S.K. participated in RT-PCR and data analysis and C.-X.D. designed the strategy and wrote the paper.

Corresponding author

Correspondence to Chu-Xia Deng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–3 (PDF 1225 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Shukla, V., Coumoul, X., Wang, RH. et al. RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis. Nat Genet 39, 1145–1150 (2007). https://doi.org/10.1038/ng2096

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng2096

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing