Skip to main content

The conditioned medium from a stable human GDF3-expressing CHO cell line, induces the differentiation of PC12 cells

Molecular and Cellular Biochemistry volume 359, pages 115–123 (2012)Cite this article

Abstract

Members of the transforming growth factor-β (TGF-β) superfamily have significant roles in the regulation of a wide variety of physiological processes. In our present work, phylogenetic tree analysis showed that human GDF3 (Growth and differentiation factor 3) and human GDF1 formed a subgroup of closely related molecules. Through quantitative real-time PCR analysis in different human tissues, GDF1 and GDF3 expression level had a big difference in brain. GDF3 could activate downstream signaling through associating with ALK7 (Activin receptor-like kinase 7) in a Cripto-dependent fashion. A CHO cell line stably transfected with the encoding sequence of GDF3, named CHO-GDF3, was established. Western blotting analysis demonstrated that GDF3 protein could be secreted into the medium from CHO cells and immunofluorescence experiment showed that GDF3 was mainly distributed in cytoplasm of the stable cell line, the primary hippocampal neurons, and brain tissues. Furthermore, the conditioned medium from CHO-GDF3 could reduce PC12 cell growth and induce cell differentiation. All these findings bring new insights into the functional study of GDF3.

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

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Abbreviations

TGF-β superfamily:

Transforming growth factor-β superfamily

GDF3:

Growth and differentiation factor 3

ALK7:

Activin receptor like-kinase 7

BMP:

Bone morphogenetic protein

β2-MG:

β2-macroglobulin

References

  1. Yingling JM, Blanchard KL, Sawyer JS (2004) Development of TGF-beta signalling inhibitors for cancer therapy. Nat Rev Drug Discov 3:1011–1022

    PubMed  Article  CAS  Google Scholar 

  2. de Caestecker M (2004) The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev 15:1–11

    PubMed  Article  Google Scholar 

  3. Constam DB, Robertson EJ (1999) Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases. J Cell Biol 144:139–149

    PubMed  Article  CAS  Google Scholar 

  4. Molloy SS, Bresnahan PA, Leppla SH, Klimpel KR, Thomas G (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402

    PubMed  CAS  Google Scholar 

  5. Creemers JW, Groot Kormelink PJ, Roebroek AJ, Nakayama K, Van de Ven WJ (1993) Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4. FEBS Lett 336:65–69

    PubMed  Article  CAS  Google Scholar 

  6. Hosaka M, Murakami K, Nakayama K (1994) PACE4 is a ubiquitous endoprotease that has similar but not identical substrate specificity to other KEX2-like processing endoproteases. Biomed Res 16:383–390

    Google Scholar 

  7. Herpin A, Lelong C, Favrel P (2004) Transforming growth factor-beta-related proteins: an ancestral and widespread superfamily of cytokines in metazoans. Dev Comp Immunol 28:461–485

    PubMed  Article  CAS  Google Scholar 

  8. Daopin S, Piez KA, Ogawa Y, Davies DR (1992) Crystal structure of transforming growth factor-beta 2: an unusual fold for the superfamily. Science 257:369–373

    PubMed  Article  CAS  Google Scholar 

  9. Schlunegger MP, Grutter MG (1992) An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2. Nature 358:430–434

    PubMed  Article  CAS  Google Scholar 

  10. Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K (2002) Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells 7:1191–1204

    PubMed  Article  CAS  Google Scholar 

  11. McPherron AC, Lee SJ (1993) GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines. J Biol Chem 268:3444–3449

    PubMed  CAS  Google Scholar 

  12. Wang W, Yang Y, Meng Y, Shi Y (2004) GDF-3 is an adipogenic cytokine under high fat dietary condition. Biochem Biophys Res Commun 321:1024–1031

    PubMed  Article  CAS  Google Scholar 

  13. Andersson O, Korach-Andre M, Reissmann E, Ibáñez CF, Bertolino P (2008) Growth/differentiation factor 3 signals through ALK7 and regulates accumulation of adipose tissue and diet-induced obesity. Proc Natl Acad Sci 105:7252–7256

    PubMed  Article  CAS  Google Scholar 

  14. Shen JJ, Huang L, Li L, Jorgez C, Matzuk MM, Brown CW (2009) Deficiency of growth differentiation factor 3 protects against diet-induced obesity by selectively acting on white adipose. Mol Endocrinol 23:113–123

    PubMed  Article  CAS  Google Scholar 

  15. Clark AT, Rodriguez RT, Bodnar MS, Abeyta MJ, Cedars MI, Turek PJ, Firpo MT, Reijo Pera RA (2004) Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma. Stem Cells 22:169–179

    PubMed  Article  CAS  Google Scholar 

  16. Ezeh UI, Turek PJ, Reijo RA, Clark AT (2005) Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma. Cancer 104:2255–2265

    PubMed  Article  CAS  Google Scholar 

  17. Andersson O, Bertolino P, Ibáñez CF (2007) Distinct and cooperative roles of mammalian Vg1 homologs GDF1 and GDF3 during early embryonic development. Dev Biol 11:500–511

    Article  Google Scholar 

  18. Chen C, Ware SM, Sato A, Houston-Hawkins DE, Habas R, Matzuk MM, Shen MM, Brown CW (2006) The Vg1-related protein Gdf3 acts in a nodal signaling pathway in the pre-gastrulation mouse embryo. Development 133:319–329

    PubMed  Article  CAS  Google Scholar 

  19. Levine AJ, Brivanlou AH (2006) GDF3, a BMP inhibitor, regulates cell fate in stem cells and early embryos. Development 133:209–216

    PubMed  Article  CAS  Google Scholar 

  20. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  21. Wu Y, Zuo J, Ji G, Saiyin H, Liu X, Yin F, Cao N, Wen Y, Li JJ, Yu L (2009) Proapoptotic function of integrin beta(3) in human hepatocellular carcinoma cells. Clin Cancer Res 15:60–69

    PubMed  Article  CAS  Google Scholar 

  22. Hexige S, Guo J, Ma L, Sun Y, Liu X, Ma L, Yan X, Li Z, Yu L (2005) Expression pattern of growth/differentiation factor 3 in human and murine cerebral cortex, hippocampus as well as cerebellum. Neurosci Lett 389:83–87

    PubMed  Article  CAS  Google Scholar 

  23. Jörnvall H, Blokzijl A, ten Dijke P, Ibáñez CF (2001) The orphan receptor serine/threonine kinase ALK7 signals arrest of proliferation and morphological differentiation in a neuronal cell line. J Biol Chem 276:5140–5146

    PubMed  Article  Google Scholar 

  24. Levine AJ, Levine ZJ, Brivanlou AH (2009) GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses. Dev Biol 325:43–48

    PubMed  Article  CAS  Google Scholar 

  25. Ge G, Hopkins DR, Ho WB, Greenspan DS (2005) GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells. Mol Cell Biol 25:5846–5858

    PubMed  Article  CAS  Google Scholar 

  26. Yang YJ, Lee HJ, Choi DH, Huang HS, Lim SC, Lee MK (2008) Effect of scoparone on neurite outgrowth in PC12 cells. Neurosci Lett 440:14–18

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the National 973 program of China (2004CB518605), the National 863 project of China (2006AA020501), the National Key Sci-Tech Special Project of China (2008ZX10002-020), the Project of the Shanghai Municipal Science and Technology Commission (03dz14086), and the National Natural Science foundation of China (30024001, 30771188).

Author information

Affiliations

  1. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, People’s Republic of China

    Qiang Li, Xianghua Liu, Yanhua Wu, Jian An, Saiyin Hexige, Yichen Ling, Mingjun Zhang, Xianmei Yang & Long Yu

Authors
  1. Qiang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xianghua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yanhua Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jian An
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saiyin Hexige
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yichen Ling
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mingjun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xianmei Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Long Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Qiang Li or Long Yu.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, Q., Liu, X., Wu, Y. et al. The conditioned medium from a stable human GDF3-expressing CHO cell line, induces the differentiation of PC12 cells. Mol Cell Biochem 359, 115–123 (2012). https://doi.org/10.1007/s11010-011-1005-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-011-1005-0

Keywords

  • TGF-β superfamily
  • GDF3
  • ALK7
  • PC12
  • Differentiation
  • Proliferation