, Volume 63, Issue 2, pp 101–109 | Cite as

An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines

  • Satoshi Abe
  • Ryuzo Sasaki
  • Seiji MasudaEmail author
JAACT Special Issue


Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.


Erythropoietin Erythropoietin receptor JAK2 Apoptosis Antibody Phosphorylation Proliferation Fragmentation 





EPO receptor


Soluble form of EPOR


Colony-forming unit erythroid




Minimum essential medium


Fetal calf serum




3-(4,5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide


Janus family of nonreceptor-type protein tyrosine kinase


Signal transducers and activators of transcription



This work was supported by Grants-in aid from the Ministry of education, Science and Culture of Japan and from Takeda Science Foundation. We are grateful to Naoko Fujiwara for the fruitful discussion and critical comments.


  1. D’Andrea AD, Lodish HF, Wong GG, (1989) Expression cloning of the murine erythropoietin receptor. Cell 57:277–285CrossRefGoogle Scholar
  2. Damen JE, Wakao H, Miyajima A, Krosl J, Humphries RK, Cutler RL, Krystal G (1995) Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation. EMBO J 14:5557–5568Google Scholar
  3. Ebie AZ, Fleming KG (2007) Dimerization of the erythropoietin receptor transmembrane domain in micelles. J Mol Biol 366:517–524CrossRefGoogle Scholar
  4. Gobert S, Chretien S, Gouilleux F, Muller O, Pallard C, Dusanter-Fourt I, Groner B, Lacombe C, Gisselbrecht S, Mayeux P (1996) Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation. EMBO J 15:2434–2441Google Scholar
  5. Gregoli PA, Bondurant MC (1997) The roles of Bcl-X (L) and apopain in the control of erythropoiesis by erythropoietin. Blood 90:630–640Google Scholar
  6. Jelkmann W (2004) Molecular biology of erythropoietin. Intern Med 43:649–659CrossRefGoogle Scholar
  7. Jelkmann W (2007) Erythropoietin after a century of research: younger than ever. Eur J Haematol 78:183–205CrossRefGoogle Scholar
  8. Jiao H, Berrada K, Yang W, Tabrizi M, Platanias LC, Yi T (1996) Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1. Mol Cell Biol 16:6985–6992Google Scholar
  9. Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF (1995) Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80:729–738CrossRefGoogle Scholar
  10. Klingmuller U, Bergelson S, Hsiao JG, Lodish HF(1996) Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5. Proc Natl Acad Sci USA 93:8324–8328CrossRefGoogle Scholar
  11. Livnah O, Johnson DL, Stura EA, Farrell FX, Barbone FP, You Y, Liu KD, Goldsmith MA, He W, Krause CD, Pestka S, Jolliffe LK (1998) An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation. Nat Struct Biol 5:993–1004CrossRefGoogle Scholar
  12. Livnah O, Stura EA, Middleton SA, Johnson DL, Jolliffe LK, Wilson IA (1999) Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 283:987–990CrossRefGoogle Scholar
  13. Masuda S, Nagao M, Takahata K, Konishi Y, Gallyas F Jr, Tabira T, Sasaki R (1993) Functional erythropoietin receptor of the cells with neural characteristics. Comparison with receptor properties of erythroid cells. J Biol Chem 268:11208–11216Google Scholar
  14. Nagao M, Masuda S, Abe S, Ueda M, Sasaki R (1992) Production and ligand-binding characteristics of the soluble form of murine erythropoietin receptor. Biochem Biophys Res Commun 188:888–897CrossRefGoogle Scholar
  15. Philo JS, Aoki KH, Arakawa T, Narhi LO, Wen J (1996) Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction. Biochemistry 35:1681–1691CrossRefGoogle Scholar
  16. Remy I, Wilson IA, Michnick SW (1999) Erythropoietin receptor activation by a ligand-induced conformation change. Science 283:990–993CrossRefGoogle Scholar
  17. Sasaki R (2003) Pleiotropic functions of erythropoietin. Intern Med 42:142–149CrossRefGoogle Scholar
  18. Sasaki R, Masuda S, Nagao M (2000) Erythropoietin: multiple physiological functions and regulation of biosynthesis. Biosci Biotechnol Biochem 64:1775–1793CrossRefGoogle Scholar
  19. Sasaki R, Masuda S, Nagao M (2001) Pleiotropic functions and tissue-specific expression of erythropoietin. News Physiol Sci 16:110–113Google Scholar
  20. Seubert N, Royer Y, Staerk J, Kubatzky KF, Moucadel V, Krishnakumar S, Smith SO, Constantinescu SN (2003) Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer. Mol Cell 12:1239–1250CrossRefGoogle Scholar
  21. Silva M, Benito A, Sanz C, Prosper F, Ekhterae D, Nuñez G, Fernandez-Luna JL (1999) Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 274:22165–22169CrossRefGoogle Scholar
  22. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF (1999) Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98:181–191CrossRefGoogle Scholar
  23. Syed RS, Reid SW, Li C, Cheetham JC, Aoki KH, Liu B, Zhan H, Osslund TD, Chirino AJ, Zhang J, Finer-Moore J, Elliott S, Sitney K, Katz BA, Matthews DJ, Wendoloski JJ, Egrie J, Stroud RM (1998) Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature 395:511–516CrossRefGoogle Scholar
  24. Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN (1993) JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74:227–236CrossRefGoogle Scholar
  25. Yamaguchi K, Akai K, Kawanishi G, Ueda M, Masuda S, Sasaki R (1991) Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties. J Biol Chem 266:20434–20439Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Department of Food Science and Technology, Faculty of AgricultureKyoto UniversityKyotoJapan
  2. 2.Graduate School of BiostudiesKyoto UniversityKyotoJapan

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