, Volume 24, Issue 5, pp 847–856 | Cite as

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway

  • Kei-ichi Nakajima
  • Yosuke Kanno
  • Masato Nakamura
  • Xiao-Dong Gao
  • Asami Kawamura
  • Fumiaki Itoh
  • Akira Ishisaki


Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.


FGF2 Lactoferrin Osteoblasts p44/p42 MAP kinase VEGF 





Bovine LF


Vascular endothelial growth factor


Fibroblast growth factor


Mitogen-activated protein


Fetal calf serum


Human LF


  1. Anderson BF, Baker HM, Norris GE, Rice DW, Baker EN (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J Mol Biol 209:711–734PubMedCrossRefGoogle Scholar
  2. Asahara T, Bauters C, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM (1995) Synergistic effect of vascular endothelial growth-factor and basic fibroblast growth-factor on angiogenesis in vivo. Circulation 92:365–371Google Scholar
  3. Berra E, Pages G, Pouyssegur J (2000) MAP kinases and hypoxia in the control of VEGF expression. Cancer Metastasis Rev 19:139–145PubMedCrossRefGoogle Scholar
  4. Blais A, Malet A, Mikogami T, Martin-Rouas C, Tome D (2009) Oral bovine lactoferrin improves bone status of ovariectomized mice. Am J Physiol Endocrinol Metab 296:E1281–E1288PubMedCrossRefGoogle Scholar
  5. Caccavo D, Pellegrino NM, Altamura M, Rigon A, Amati L, Amoroso A, Jirillo E (2002) Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application. J Endotoxin Res 8:403–417PubMedGoogle Scholar
  6. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435–439PubMedCrossRefGoogle Scholar
  7. Cheng T, Cao W, Wen R, Steinberg RH, LaVail MM (1998) Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Muller cells. Invest Ophthalmol Vis Sci 39:581–591PubMedGoogle Scholar
  8. Chua CC, Hamdy RC, Chua BH (2000) Mechanism of transforming growth factor-beta1-induced expression of vascular endothelial growth factor in murine osteoblastic MC3T3-E1 cells. Biochim Biophys Acta 1497:69–76PubMedCrossRefGoogle Scholar
  9. Cornish J, Callon KE, Naot D, Palmano KP, Banovic T, Bava U, Watson M, Lin JM, Tong PC, Chen Q, Chan VA, Reid HE, Fazzalari N, Baker HM, Baker EN, Haggarty NW, Grey AB, Reid IR (2004) Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145:4366–4374PubMedCrossRefGoogle Scholar
  10. Erlebacher A, Filvaroff EH, Gitelman SE, Derynck R (1995) Toward a molecular understanding of skeletal development. Cell 80:371–378PubMedCrossRefGoogle Scholar
  11. Ferrara N (1999) Molecular and biological properties of vascular endothelial growth factor. J Mol Med 77:527–543PubMedCrossRefGoogle Scholar
  12. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439–442PubMedCrossRefGoogle Scholar
  13. Fiorelli G, Orlando C, Benvenuti S, Franceschelli F, Bianchi S, Pioli P, Tanini A, Serio M, Bartucci F, Brandi ML (1994) Characterization, regulation, and function of specific cell-membrane receptors for insulin-like growth-factor-I on bone endothelial-cells. J Bone Miner Res 9:329–337PubMedCrossRefGoogle Scholar
  14. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16:4604–4613PubMedGoogle Scholar
  15. Friesel R, Maciag T (1999) Fibroblast growth factor prototype release and fibroblast growth factor receptor signaling. Thromb Haemost 82:748–754PubMedGoogle Scholar
  16. Füreder W, Krauth MT, Sperr WR, Sonneck K, Simonitsch-Klupp I, Müllauer L, Willmann M, Horny HP, Valent P (2006) Evaluation of angiogenesis and vascular endothelial growth factor expression in the bone marrow of patients with aplastic anemia. Am J Pathol 168:123–130PubMedCrossRefGoogle Scholar
  17. Globus RK, Plouet J, Gospodarowicz D (1989) Cultured bovine bone cells synthesize basic fibroblast growth factor and store it in their extracellular matrix. Endocrinology 124:1539–1547PubMedCrossRefGoogle Scholar
  18. Goretzki L, Mueller BM (1998) Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein. Biochem J 336:381–386PubMedGoogle Scholar
  19. Goto F, Goto K, Weindel K, Folkman J (1993) Synergistic effects of vascular endothelial growth-factor and basic fibroblast growth-factor on the proliferation and cord formation of bovine capillary endothelial-cells within collagen gels. Lab Invest 69:508–517PubMedGoogle Scholar
  20. Grey A, Banovic T, Zhu Q, Watson M, Callon K, Palmano K, Ross J, Naot D, Reid IR, Cornish J (2004) The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells. Mol Endocrinol 18:2268–2278PubMedCrossRefGoogle Scholar
  21. Grey A, Zhu Q, Watson M, Callon K, Cornish J (2006) Lactoferrin potently inhibits osteoblast apoptosis, via an LRP1-independent pathway. Mol Cell Endocrinol 251:96–102PubMedCrossRefGoogle Scholar
  22. Guenther HL, Fleisch H, Sorgente N (1986) Endothelial-cells in culture synthesize a potent bone cell active mitogen. Endocrinology 119:193–201PubMedCrossRefGoogle Scholar
  23. Guo HY, Jiang L, Ibrahim SA, Zhang L, Zhang H, Zhang M, Ren FZ (2009) Orally administered lactoferrin preserves bone mass and microarchitecture in ovariectomized rats. J Nutr 139:958–964PubMedCrossRefGoogle Scholar
  24. Hasegawa K, Motsuchi W, Tanaka S, Dosako S (1994) Inhibition with lactoferrin of in vitro infection with human herpes virus. Jpn J Med Sci Biol 47:73–85PubMedGoogle Scholar
  25. Hurley MM, Abreu C, Gronowicz G, Kawaguchi H, Lorenzo J (1994) Expression and regulation of basic fibroblast growth-factor messenger-RNA levels in mouse osteoblastic MC3T3-E1 cells. J Biol Chem 269:9392–9396PubMedGoogle Scholar
  26. Hurley MM, Tetradis S, Huang YF, Hock J, Kream BE, Raisz LG, Sabbieti MG (1999) Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells. J Bone Miner Res 14:776–783PubMedCrossRefGoogle Scholar
  27. Ji ZS, Mahley RW (1994) Lactoferrin binding to heparin sulfate proteoglycans and the LDL receptor-related protein. Further evidence supporting the importance of direct binding of remnant lipoproteins to HSPG. Arterioscler Thromb 14:2025–2031PubMedCrossRefGoogle Scholar
  28. Kim CW, Son KN, Choi SY, Kim JY (2006) Human lactoferrin upregulates expression of KDR/Flk-1 and stimulates VEGF-A-mediated endothelial cell proliferation and migration. FEBS Lett 580:4332–4336PubMedCrossRefGoogle Scholar
  29. Li XH, Zhou X, Zeng S, Ye F, Yun JL, Huang TG, Li H, Li YM (2008) Effects of intramyocardial injection of platelet-rich plasma on the healing process after myocardial infarction. Coron Artery Dis 19:363–370PubMedCrossRefGoogle Scholar
  30. Lorget F, Clough J, Oliveira M, Daury MC, Sabokbar A, Offord E (2002) Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity. Biochem Biophys Res Commun 296:261–266PubMedCrossRefGoogle Scholar
  31. Neels JG, van Den Berg BM, Lookene A, Olivecrona G, Pannekoek H, van Zonneveld AJ (1999) The second and fourth cluster of class a cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties. J Biol Chem 274:31305–31311PubMedCrossRefGoogle Scholar
  32. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z (1999) Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 13:9–22PubMedGoogle Scholar
  33. Nijweide PJ, Burger EH, Feyen JHM (1986) Cells of bone—proliferation, differentiation, and hormonal-regulation. Physiol Rev 66:855–886PubMedGoogle Scholar
  34. Norrby K (2004) Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo. J Vasc Res 41:293–304PubMedCrossRefGoogle Scholar
  35. Norrby K, Mattsby-Baltzer I, Innocenti M, Tuneberg S (2001) Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat. Int J Cancer 91:236240CrossRefGoogle Scholar
  36. Paesano R, Berlutti F, Pietropaoli M, Pantanella F, Pacifici E, Goolsbee W, Valenti P (2010) Lactoferrin efficacy versus ferrous sulfate in curing iron deficiency and iron deficiency anemia in pregnant women. Biometals 23:411–417PubMedCrossRefGoogle Scholar
  37. Pepper MS, Ferrara N, Orci L, Montesano R (1992) Potent synergism between vascular endothelial growth-factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 189:824–831PubMedCrossRefGoogle Scholar
  38. Pierce A, Colavizza D, Benaissa M, Maes P, Tartar A, Montreuil J, Spik G (1991) Molecular cloning and sequence analysis of bovine lactotransferrin. Eur J Biochem 196:177–184PubMedCrossRefGoogle Scholar
  39. Powers CJ, McLeskey SW, Wellstein A (2000) Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7:165–197PubMedCrossRefGoogle Scholar
  40. Shima DT, Deutsch U, D’Amore PA (1995) Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability. FEBS Lett 370:203–208PubMedCrossRefGoogle Scholar
  41. Shimamura M, Yamamoto Y, Ashino H, Oikawa T, Hazato T, Tsuda H, Iigo M (2004) Bovine lactoferrin inhibits tumor-induced angiogenesis. Int J Cancer 111:111–116PubMedCrossRefGoogle Scholar
  42. Steijns JM, van Hooijdonk ACM (2000) Occurrence structure, biochemical properties and technological characteristics of lactoferrin. Br J Nutr 84:S11–S17PubMedCrossRefGoogle Scholar
  43. Street J, Lenehan B (2009) Vascular endothelial growth factor regulates osteoblast survival—evidence for an autocrine feedback mechanism. J Orthop Surg Res 4:19PubMedCrossRefGoogle Scholar
  44. Suda T, Jimi E, Nakamura I, Takahashi N (1997) Role of 1 alpha, 25-dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 282:223–235PubMedCrossRefGoogle Scholar
  45. Takayama Y, Mizumachi K (2008) Effect of bovine lactoferrin on extracellular matrix calcification by human osteoblast-like cells. Biosci Biotechnol Biochem 72:226–230PubMedCrossRefGoogle Scholar
  46. Takayama Y, Mizumachi K (2009) Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells. J Biosci Bioeng 107:191–195PubMedCrossRefGoogle Scholar
  47. Tsuda H, Sekine K, Fujita K, Iigo M (2002) Cancer prevention by bovine lactoferrin and underlying mechanisms—a review of experimental and clinical studies. Biochem Cell Biol 80:131–136PubMedCrossRefGoogle Scholar
  48. Vash B, Phung N, Zein S, DeCamp D (1998) Three complement-type repeats of the low-density lipoprotein receptor-related protein define a common binding site for RAP, PAI-1, and lactoferrin. Blood 92:3277–3285PubMedGoogle Scholar
  49. Villanueva JE, Nimni ME (1990) Promotion of calvarial cell osteogenesis by endothelial-cells. J Bone Miner Res 5:733–739PubMedCrossRefGoogle Scholar
  50. Villars F, Bordenave L, Bareille R, Amedee J (2000) Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF? J Cell Biochem 79:672–685PubMedCrossRefGoogle Scholar
  51. Willnow TE, Goldstein JL, Orth K, Brown MS, Herz J (1992) Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. J Biol Chem 267:26172–26180PubMedGoogle Scholar
  52. Yamauchi K, Wakabayashi H, Shin K, Takase M (2006) Bovine Lactoferrin: benefits and mechanism of action against infections. Biochem Cell Biol 84:291–296PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Kei-ichi Nakajima
    • 1
  • Yosuke Kanno
    • 2
  • Masato Nakamura
    • 1
  • Xiao-Dong Gao
    • 3
  • Asami Kawamura
    • 1
  • Fumiaki Itoh
    • 1
  • Akira Ishisaki
    • 4
  1. 1.National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research OrganizationSapporoJapan
  2. 2.Department of Clinical Pathological Biochemistry, Faculty of Pharmaceutical ScienceDoshisha Women’s College of Liberal ArtsKyotoJapan
  3. 3.Frontier Research Center for Post-Genomic Science and TechnologyHokkaido UniversitySapporoJapan
  4. 4.Department of BiochemistryIwate Medical University School of DentistryMoriokaJapan

Personalised recommendations