Skip to main content
Download PDF

Osteoblast differentiation of NIH3T3 fibroblasts is associated with changes in the IGF-I/IGFBP expression pattern

Cellular & Molecular Biology Letters volume 11, pages461–474(2006)Cite this article

Abstract

Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are essential regulators for osteoblast proliferation and differentiation. It has been reported that Dexamethasone (Dex), an active glucocorticoid (GC) analogue, synergizes the stimulatory effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast differentiation in the mouse fibroblastic cell line NIH3T3. I investigated whether this stimulatory effect is associated with changes in the expression pattern of the IGF/IGFBP system. Quantitative real-time PCR technology was used to quantify the gene expression levels of the IGF-system during osteoblast differentiation and in response to 1,25(OH)2D3 or Dex alone under serum-containing and serum-free culture conditions. Interestingly, NIH3T3 was shown to express high mRNA levels of IGF-I, IGF-II and IGFBP-5, and low levels of both IGFBP-2 and-6. During osteoblast differentiation (days 6-12), IGF-I mRNA was repressed by more than 60%, while the transcript of IGFBP-5 was markedly up-regulated, by more than 50-fold. Similarly, treatment with Dex alone resulted in a dose-and time-dependent increase in the expression of IGFBP-5 and a decrease in IGF-I mRNA. Treatment with 1,25(OH)2D3 alone increased the mRNA levels of IGF-I and IGFBP-6 by around 4-and 7-fold, respectively, in a dose-and time-dependent manner. In conclusion, my data demonstrated that osteoblast differentiation of NIH3T3 is associated with changes in the expression pattern of IGFs/IGFBPs, which are regulated by glucocorticoid in the presence of 1,25(OH)2D3. Modulation of the IGF/IGFBP levels by glucocorticoid might suggest important roles for the IGF-system in mediating the osteoblast differentiation of the NIH3T3 cell line.

Abbreviations

ALP:

alkaline phosphatase

BMP-2:

Bone morphogenetic protein-2

Cbfa1/Runx2:

Core binding factor

runt domain:

alfa subunit 1

Dex:

Dexamethasone

GC:

glucocorticoid

IGFBPs:

insulin like growth factor binding proteins

IGFs:

insulin like growth factors

MSC:

mesenchymal stem cells

OC:

osteocalcin

OPN:

osteopontein

RT:

reverse transcriptase

References

  1. 1.

    Kassem, M., Kristiansen, M. and Abdallah, B.M. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin. Pharmacol. Toxicol. 95 (2004) 209–214.

    PubMed  CAS  Article  Google Scholar 

  2. 2.

    Langdahl, B.L., Kassem, M., Moller, M.K. and Eriksen, E.F. The effects of IGF-I and IGF-II on proliferation and differentiation of human osteoblasts and interactions with growth hormone. Eur. J. Clin. Invest. 28 (1998) 176–183.

    PubMed  CAS  Article  Google Scholar 

  3. 3.

    Rajaram, S., Baylink, D.J. and Mohan, S. Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr. Rev. 18 (1997) 801–831.

    PubMed  CAS  Article  Google Scholar 

  4. 4.

    Mohan, S. and Baylink, D.J. IGF-binding proteins are multifunctional and act via IGF-dependent and-independent mechanisms. J. Endocrinol. 175 (2002) 19–31.

    PubMed  CAS  Article  Google Scholar 

  5. 5.

    Robson, H., Siebler, T., Shalet, S.M. and Williams, G.R. Interactions between GH, IGF-I, glucocorticoids, and thyroid hormones during skeletal growth. Pediatr. Res. 52 (2002) 137–147.

    PubMed  CAS  Article  Google Scholar 

  6. 6.

    Eijken, M., Koedam, M., van Driel, M., Buurman, C.J., Pols, H.A. and van Leeuwen, J.P. The essential role of glucocorticoids for proper human osteoblast differentiation and matrix mineralization. Mol. Cell Endocrinol. 248 (2006) 87–93.

    PubMed  CAS  Article  Google Scholar 

  7. 7.

    Delany, A.M. and Canalis, E. Transcriptional repression of insulin-like growth factor I by glucocorticoids in rat bone cells. Endocrinology 136 (1995) 4776–4781.

    PubMed  CAS  Article  Google Scholar 

  8. 8.

    Cheng, S.L., Zhang, S.F., Mohan, S., Lecanda, F., Fausto, A., Hunt, A.H., Canalis, E. and Avioli, L.V. Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone. J. Cell Biochem. 71 (1998) 449–458.

    PubMed  CAS  Article  Google Scholar 

  9. 9.

    Gurlek, A., Pittelkow, M.R. and Kumar, R. Modulation of growth factor/cytokine synthesis and signaling by 1alpha,25-dihydroxyvitamin D(3): implications in cell growth and differentiation. Endocr. Rev. 23 (2002) 763–786.

    PubMed  CAS  Article  Google Scholar 

  10. 10.

    Kveiborg, M., Flyvbjerg, A., Eriksen, E.F. and Kassem, M. 1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2,-3 and-4 in human bone marrow stromal cells. Eur. J. Endocrinol. 144 (2001) 549–557.

    PubMed  CAS  Article  Google Scholar 

  11. 11.

    Nakao, Y., Hilliker, S., Baylink, D.J. and Mohan, S. Studies on the regulation of insulin-like growth factor binding protein 3 secretion in human osteosarcoma cells in vitro. J. Bone Miner. Res. 9 (1994) 865–872.

    PubMed  CAS  Article  Google Scholar 

  12. 12.

    Shui, C. and Scutt, A.M. Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro. J. Cell Physiol. 193 (2002) 164–172.

    PubMed  CAS  Article  Google Scholar 

  13. 13.

    Wang, E.A., Israel, D.I., Kelly, S. and Luxenberg, D.P. Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors 9 (1993) 57–71.

    PubMed  CAS  Google Scholar 

  14. 14.

    Xiao, Z.S., Hinson, T.K. and Quarles, L.D. Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells. J. Cell Biochem. 74 (1999) 596–605.

    PubMed  CAS  Article  Google Scholar 

  15. 15.

    Abdallah, B.M., Haack-Sorensen, M., Fink, T. and Kassem, M. Inhibition of osteoblast differentiation but not adipocyte differentiation of mesenchymal stem cells by sera obtained from aged females. Bone 39 (2006) 181–188.

    PubMed  Article  Google Scholar 

  16. 16.

    Koch, H., Jadlowiec, J.A. and Campbell, P.G. Insulin-like growth factor-I induces early osteoblast gene expression in human mesenchymal stem cells. Stem Cells Dev. 14 (2005) 621–631.

    PubMed  CAS  Article  Google Scholar 

  17. 17.

    Barnes, P.J. Corticosteroids: the drugs to beat. Eur. J. Pharmacol. 533 (2006) 2–14.

    PubMed  CAS  Article  Google Scholar 

  18. 18.

    Canalis, E., Bilezikian, J.P., Angeli, A. and Giustina, A. Perspectives on glucocorticoid-induced osteoporosis. Bone 34 (2004) 593–598.

    PubMed  CAS  Article  Google Scholar 

  19. 19.

    Smith, E., Coetzee, G.A. and Frenkel, B. Glucocorticoids inhibit cell cycle progression in differentiating osteoblasts via glycogen synthase kinase-3beta. J. Biol. Chem. 277 (2002) 18191–18197.

    PubMed  CAS  Article  Google Scholar 

  20. 20.

    Jorgensen, N.R., Henriksen, Z., Sorensen, O.H. and Civitelli, R. Dexamethasone, BMP-2and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids 69 (2004) 219–226.

    PubMed  CAS  Article  Google Scholar 

  21. 21.

    Minuto, F., Palermo, C., Arvigo, M. and Barreca, A.M. The IGF system and bone. J. Endocrinol. Invest. 28 (2005) 8–10.

    PubMed  CAS  Google Scholar 

  22. 22.

    Chen, T.L., Mallory, J.B. and Hintz, R.L. Dexamethasone and 1,25(OH)2 vitamin D3 modulate the synthesis of insulin-like growth factor-I insteoblast-like cells. Calcif. Tissue Int. 48 (1991) 278–282.

    PubMed  CAS  Google Scholar 

  23. 23.

    Chevalley, T., Strong, D.D., Mohan, S., Baylink, D. and Linkhart, T.A. Evidence for a role for insulin-like growth factor binding proteins in glucocorticoid inhibition of normal human osteoblast-like cell proliferation. Eur. J. Endocrinol. 134 (1996) 591–601.

    PubMed  CAS  Article  Google Scholar 

  24. 24.

    Conover, C.A., Clarkson, J.T. and Bale, L.K. Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF-binding protein expression in fibroblasts. Endocrinology 136 (1995) 1403–1410.

    PubMed  CAS  Article  Google Scholar 

  25. 25.

    Fielder, P.J., Tauber, J.P., Wilson, K.F., Pham, H.M. and Rosenfeld, R.G. Insulin-like growth factors (IGFs) stimulate and dexamethasone inhibits IGF binding protein (BP)-5 expression in a mouse pituitary cell line. Growth Regul. 3 (1993) 226–234.

    PubMed  CAS  Google Scholar 

  26. 26.

    Firth, S.M. and Baxter, R.C. Cellular actions of the insulin-like growth factor binding proteins. Endocr. Rev. 23 (2002) 824–854.

    PubMed  CAS  Article  Google Scholar 

  27. 27.

    Miyakoshi, N., Richman, C., Kasukawa, Y., Linkhart, T.A., Baylink, D.J. and Mohan, S. Evidence that IGF-binding protein-5 functions as a growth factor. J. Clin. Invest. 107 (2001) 73–81.

    PubMed  CAS  Article  Google Scholar 

  28. 28.

    Durant, D., Pereira, R.M. and Canalis, E. Overexpression of insulin-like growth factor binding protein-5 decreases osteoblastic function in vitro. Bone 35 (2004) 1256–1262.

    PubMed  CAS  Article  Google Scholar 

  29. 29.

    Jia, D. and Heersche, J.N. Expression of insulin-like growth factor system constituents in differentiating rat osteoblastic cell populations. Growth Horm. IGF. Res. 12 (2002) 399–410.

    PubMed  CAS  Article  Google Scholar 

  30. 30.

    Chen, T.L., Chang, L.Y., Bates, R.L. and Perlman, A.J. Dexamethasone and 1,25-dihydroxyvitamin D3 modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cell cultures. Endocrinology 128 (1991) 73–80.

    PubMed  CAS  Google Scholar 

  31. 31.

    Chenu, C., Valentin-Opran, A., Chavassieux, P., Saez, S., Meunier, P.J. and Delmas, P.D. Insulin like growth factor I hormonal regulation by growth hormone and by 1,25(OH)2D3 and activity on human osteoblast-like cells in short-term cultures. Bone 11 (1990) 81–86.

    PubMed  CAS  Article  Google Scholar 

  32. 32.

    Linkhart, T.A. and Keffer, M.J. Differential regulation of insulin-like growth factor-I (IGF-I) and IGF-II release from cultured neonatal mouse calvaria by parathyroid hormone, transforming growth factor-betaand 1,25-dihydroxyvitamin D3. Endocrinology 128 (1991) 1511–1518.

    PubMed  CAS  Article  Google Scholar 

  33. 33.

    Schmid, C., Schlapfer, I., Gosteli-Peter, M.A., Hauri, C., Froesch, E.R. and Zapf, J. 1 alpha,25-dihydroxyvitamin D3 increases IGF binding protein-5 expression in cultured osteoblasts. FEBS Lett. 392 (1996) 21–24.

    PubMed  CAS  Article  Google Scholar 

  34. 34.

    Kim, E.J., Schaffer, B.S., Kang, Y.H., Macdonald, R.G. and Park, J.H. Decreased production of insulin-like growth factor-binding protein (IGFBP)-6 by transfection of colon cancer cells with an antisense IGFBP-6 cDNA construct leads to stimulation of cell proliferation. J. Gastroenterol. Hepatol. 17 (2002) 563–570.

    PubMed  CAS  Article  Google Scholar 

  35. 35.

    Schmid, C. Insulin-like growth factors. Cell Biol. Int. 19 (1995) 445–457.

    PubMed  CAS  Article  Google Scholar 

  36. 36.

    Yan, T., Wergedal, J., Zhou, Y., Mohan, S., Baylink, D.J. and Strong, D.D. Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6. Growth Horm. IGF. Res. 11 (2001) 368–377.

    PubMed  CAS  Article  Google Scholar 

  37. 37.

    Bach, L.A. IGFBP-6 five years on; not so ‘forgotten’? Growth Horm. IGF. Res. 15 (2005) 185–192.

    PubMed  CAS  Article  Google Scholar 

Download references

Author information

Author notes

  1. Basem M. Abdallah

    Present address: Department of Endocrinology and Metabolism, University Hospital of Odense, Medical Biotechnology Center, SDU, DK-5000, Odense C, Denmark

Affiliations

  1. Department of Biochemistry, Faculty of Science, Helwan University, Cairo, Egypt

    Basem M. Abdallah

Authors
  1. Basem M. Abdallah
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Abdallah, B.M. Osteoblast differentiation of NIH3T3 fibroblasts is associated with changes in the IGF-I/IGFBP expression pattern. Cell Mol Biol Lett 11, 461–474 (2006). https://doi.org/10.2478/s11658-006-0038-y

Download citation

Key words

  • IGFs/IGFBPs
  • NIH3T3
  • Gene expression
  • Osteoblast differentiation

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us