Skip to main content

Advertisement

SpringerLink
Log in

PDGF, bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways

  • Original Article
  • Published:
European Spine Journal Aims and scope Submit manuscript

Abstract

Intervertebral disc (IVD) degeneration is frequently characterized by increased cell proliferation, probably as a tissue regenerative response. Although many growth factors and their receptors have been shown to be expressed normally in the disc, and generally to be over-expressed during degeneration, not all of them have been thoroughly studied concerning their effects on IVD cell proliferation. In the present report, three potent mitogens, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) are examined regarding their capacity to induce proliferation in vitro of bovine coccygeal nucleus pulposus (NP) and annulus fibrosus (AF) cells, as well as to activate major intracellular signal transduction pathways. PDGF, bFGF and IGF-I were found to induce DNA synthesis in quiescent IVD cells in a dose-dependent manner. Maximum stimulation was induced by PDGF, while stimulation by all three factors simultaneously exceeded only slightly that caused by PDGF alone. All three growth factors were shown to phosphorylate immediately extracellular-signal regulated kinases (ERKs), while the stimulation by bFGF especially resulted in sustained ERK phosphorylation. Furthermore, all three growth factors induced phosphorylation of Akt in both Thr308 and Ser473 residues immediately after stimulation, although bFGF-induced phosphorylation was much weaker than that provoked by PDGF and IGF-I. In addition, the MEK inhibitor PD98059 and the PI 3-K inhibitor wortmannin were shown to block growth factor-induced ERK- and Akt-phosphorylation, respectively, in IVD cells. Inhibition of the MEK/ERK or the PI 3-K/Akt pathways provoked a significant decline of the proliferative effects of PDGF, bFGF or IGF-I on IVD cell cultures, while the simultaneous inhibition of both signaling pathways abolished completely the mitogenicity of these growth factors. The above effects of the three growth factors were reproduced similarly in both NP and AF cell cultures. Overall, the above results indicate that PDGF, bFGF and IGF-I stimulate the proliferation of IVD cells via the ERK and Akt signaling pathways.

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

Similar content being viewed by others

References

  1. Akeda K, An HS, Pichika R, Attawia M, Thonar EJ, Lenz ME, Uchida A, Masuda K (2006) Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine 31:959–966

    Article  PubMed  Google Scholar 

  2. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 270:27489–27494

    Article  PubMed  CAS  Google Scholar 

  3. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. Embo J 15:6541–6551

    PubMed  CAS  Google Scholar 

  4. Andersson GB (1999) Epidemiological features of chronic low-back pain. Lancet 354:581–585

    Article  PubMed  CAS  Google Scholar 

  5. Brazil DP, Hemmings BA (2001) Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci 26:657–664

    Article  PubMed  CAS  Google Scholar 

  6. Chang L, Karin M (2001) Mammalian MAP kinase signalling cascades. Nature 410:37–40

    Article  PubMed  CAS  Google Scholar 

  7. Chujo T, An HS, Akeda K, Miyamoto K, Muehleman C, Attawia M, Andersson G, Masuda K (2006) Effects of growth differentiation factor-5 on the intervertebral disc—in vitro bovine study and in vivo rabbit disc degeneration model study. Spine 31:2909–2917

    Article  PubMed  Google Scholar 

  8. Doita M, Kanatani T, Harada T, Mizuno K (1996) Immunohistologic study of the ruptured intervertebral disc of the lumbar spine. Spine 21:235–241

    Article  PubMed  CAS  Google Scholar 

  9. Duronio V, Scheid MP, Ettinger S (1998) Downstream signalling events regulated by phosphatidylinositol 3-kinase activity. Cell Signal 10:233–239

    Article  PubMed  CAS  Google Scholar 

  10. Eswarakumar VP, Lax I, Schlessinger J (2005) Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16:139–149

    Article  PubMed  CAS  Google Scholar 

  11. Ferby IM, Waga I, Hoshino M, Kume K, Shimizu T (1996) Wortmannin inhibits mitogen-activated protein kinase activation by platelet-activating factor through a mechanism independent of p85/p110-type phosphatidylinositol 3-kinase. J Biol Chem 271:11684–11688

    Article  PubMed  CAS  Google Scholar 

  12. Giannouli CC, Kletsas D (2006) TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2. Cell Signal 18:1417–1429

    Article  PubMed  CAS  Google Scholar 

  13. Gruber HE, Fisher EC Jr, Desai B, Stasky AA, Hoelscher G, Hanley EN Jr (1997) Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. Exp Cell Res 235:13–21

    Article  PubMed  CAS  Google Scholar 

  14. Gruber HE, Norton HJ, Leslie K, Hanley EN Jr (2001) Clinical and demographic prognostic indicators for human disc cell proliferation in vitro: pilot study. Spine 26:2323–2327

    Article  PubMed  CAS  Google Scholar 

  15. Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316

    PubMed  CAS  Google Scholar 

  16. Hsieh JK, Kletsas D, Clunn G, Hughes AD, Schachter M, Demoliou-Mason C (2000) p53, p21(WAF1/CIP1), and MDM2 involvement in the proliferation and apoptosis in an in vitro model of conditionally immortalized human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 20:973–981

    PubMed  CAS  Google Scholar 

  17. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B (2006) SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127:125–137

    Article  PubMed  CAS  Google Scholar 

  18. Johnson WE, Eisenstein SM, Roberts S (2001) Cell cluster formation in degenerate lumbar intervertebral discs is associated with increased disc cell proliferation. Connect Tissue Res 42:197–207

    PubMed  CAS  Google Scholar 

  19. Jones JI, Clemmons DR (1995) Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34

    Article  PubMed  CAS  Google Scholar 

  20. Le Maitre CL, Richardson SM, Baird P, Freemont AJ, Hoyland JA (2005) Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc. J Pathol 207:445–452

    Article  PubMed  CAS  Google Scholar 

  21. Lee HT, Kay EP (2003) Regulatory role of cAMP on expression of Cdk4 and p27(Kip1) by inhibiting phosphatidylinositol 3-kinase in corneal endothelial cells. Invest Ophthalmol Vis Sci 44:3816–3825

    Article  PubMed  Google Scholar 

  22. Manek NJ, MacGregor AJ (2005) Epidemiology of back disorders: prevalence, risk factors, and prognosis. Curr Opin Rheumatol 17:134–140

    Article  PubMed  Google Scholar 

  23. Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185

    Article  PubMed  CAS  Google Scholar 

  24. Masuda K, An HS (2006) Prevention of disc degeneration with growth factors. Eur Spine J 15(Suppl 15):422–432

    Article  Google Scholar 

  25. Masuda K, Takegami K, An H, Kumano F, Chiba K, Andersson GB, Schmid T, Thonar E (2003) Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads. J Orthop Res 21:922–930

    Article  PubMed  CAS  Google Scholar 

  26. Matsusaki M, Ochi M, Uchio Y, Shu N, Kurioka H, Kawasaki K, Adachi N (1998) Effects of basic fibroblast growth factor on proliferation and phenotype expression of chondrocytes embedded in collagen gel. Gen Pharmacol 31:759–764

    PubMed  CAS  Google Scholar 

  27. Melrose J, Smith S, Little CB, Kitson J, Hwa SY, Ghosh P (2002) Spatial and temporal localization of transforming growth factor-beta, fibroblast growth factor-2, and osteonectin, and identification of cells expressing alpha-smooth muscle actin in the injured anulus fibrosus: implications for extracellular matrix repair. Spine 27:1756–1764

    Article  PubMed  Google Scholar 

  28. Murakami H, Yoon ST, Attallah-Wasif ES, Tsai KJ, Fei Q, Hutton WC (2006) The expression of anabolic cytokines in intervertebral discs in age-related degeneration. Spine 31:1770–1774

    Article  PubMed  Google Scholar 

  29. Nagano T, Yonenobu K, Miyamoto S, Tohyama M, Ono K (1995) Distribution of the basic fibroblast growth factor and its receptor gene expression in normal and degenerated rat intervertebral discs. Spine 20:1972–1978

    Article  PubMed  CAS  Google Scholar 

  30. Okada-Ban M, Thiery JP, Jouanneau J (2000) Fibroblast growth factor-2. Int J Biochem Cell Biol 32:263–267

    Article  PubMed  CAS  Google Scholar 

  31. Osada R, Ohshima H, Ishihara H, Yudoh K, Sakai K, Matsui H, Tsuji H (1996) Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res 14:690–699

    Article  PubMed  CAS  Google Scholar 

  32. Oshima H, Ishihara H, Urban JP, Tsuji H (1993) The use of coccygeal discs to study intervertebral disc metabolism. J Orthop Res 11:332–338

    Article  PubMed  CAS  Google Scholar 

  33. Pratsinis H, Giannouli CC, Zervolea I, Psarras S, Stathakos D, Kletsas D (2004) Differential proliferative response of fetal and adult human skin fibroblasts to transforming growth factor-beta. Wound Repair Regen 12:374–383

    Article  PubMed  Google Scholar 

  34. Risbud MV, Fertala J, Vresilovic EJ, Albert TJ, Shapiro IM (2005) Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal. Spine 30:882–889

    Article  PubMed  Google Scholar 

  35. Risbud MV, Guttapalli A, Albert TJ, Shapiro IM (2005) Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of integrin expression and cell survival. Spine 30:2503–2509

    Article  PubMed  Google Scholar 

  36. Risbud MV, Di Martino A, Guttapalli A, Seghatoleslami R, Denaro V, Vaccaro AR, Albert TJ, Shapiro IM (2006) Toward an optimum system for intervertebral disc organ culture: TGF-beta 3 enhances nucleus pulposus and anulus fibrosus survival and function through modulation of TGF-beta-R expression and ERK signaling. Spine 31:884–890

    Article  PubMed  Google Scholar 

  37. Ross R, Raines EW, Bowen-Pope DF (1986) The biology of platelet-derived growth factor. Cell 46:155–169

    Article  PubMed  CAS  Google Scholar 

  38. Schmidt MB, Chen EH, Lynch SE (2006) A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair. Osteoarthritis Cartilage 14:403–412

    Article  PubMed  CAS  Google Scholar 

  39. Specchia N, Pagnotta A, Toesca A, Greco F (2002) Cytokines and growth factors in the protruded intervertebral disc of the lumbar spine. Eur Spine J 11:145–151

    Article  PubMed  Google Scholar 

  40. Tallquist M, Kazlauskas A (2004) PDGF signaling in cells and mice. Cytokine Growth Factor Rev 15:205–213

    Article  PubMed  CAS  Google Scholar 

  41. Thompson JP, Oegema TR Jr., Bradford DS (1991) Stimulation of mature canine intervertebral disc by growth factors. Spine 16:253–260

    Article  PubMed  CAS  Google Scholar 

  42. Tim Yoon S, Su Kim K, Li J, Soo Park J, Akamaru T, Elmer WA, Hutton WC (2003) The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro. Spine 28:1773–1780

    Article  PubMed  CAS  Google Scholar 

  43. Tolonen J, Gronblad M, Virri J, Seitsalo S, Rytomaa T, Karaharju E (1995) Basic fibroblast growth factor immunoreactivity in blood vessels and cells of disc herniations. Spine 20:271–276

    Article  PubMed  CAS  Google Scholar 

  44. Tolonen J, Gronblad M, Virri J, Seitsalo S, Rytomaa T, Karaharju EO (1997) Platelet-derived growth factor and vascular endothelial growth factor expression in disc herniation tissue: and immunohistochemical study. Eur Spine J 6:63–69

    Article  PubMed  CAS  Google Scholar 

  45. Tolonen J, Gronblad M, Vanharanta H, Virri J, Guyer RD, Rytomaa T, Karaharju EO (2006) Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor. Eur Spine J 15:588–596

    Article  PubMed  Google Scholar 

  46. Urban JP, Roberts S (2003) Degeneration of the intervertebral disc. Arthritis Res Ther 5:120–130

    Article  PubMed  Google Scholar 

  47. Walsh AJ, Bradford DS, Lotz JC (2004) In vivo growth factor treatment of degenerated intervertebral discs. Spine 29:156–163

    Article  PubMed  Google Scholar 

  48. Wang H, Kroeber M, Hanke M, Ries R, Schmid C, Poller W, Richter W (2004) Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells. J Mol Med 82:126–134

    Article  PubMed  CAS  Google Scholar 

  49. Wipf P, Halter RJ (2005) Chemistry and biology of wortmannin. Org Biomol Chem 3:2053–2061

    Article  PubMed  CAS  Google Scholar 

  50. Yoon ST (2005) Molecular therapy of the intervertebral disc. Spine J 5:280S–286S

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the European Union (“EURODISC” project, contract No QLK6-CT-2002-02582) and by a grant from the AO Fund, Switzerland (05-K68). HP was also partly supported by a postdoctoral fellowship of NCSR “Demokritos”. We would like to acknowledge Dr. Sotirios Moschovitsis, Director Veterinary of Public Health, Ministry of Rural Development and Food, Greece for helping us to obtain bovine tissue specimens.

Author information

Authors and Affiliations

  1. Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre for Scientific Research “Demokritos”, 153 10 Athens, Greece

    Harris Pratsinis & Dimitris Kletsas

Authors
  1. Harris Pratsinis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dimitris Kletsas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dimitris Kletsas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pratsinis, H., Kletsas, D. PDGF, bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways. Eur Spine J 16, 1858–1866 (2007). https://doi.org/10.1007/s00586-007-0408-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00586-007-0408-9

Keywords

Search

Navigation