Deciphering the Intracellular Signaling of Erythropoietin in Neuronal Cells

  • Murat Digicaylioglu
Part of the Methods in Molecular Biology book series (MIMB, volume 982)


The search for potential drugs to treat neurodegenerative diseases has been intense in the last two decades. Among many candidates, erythropoietin (EPO) was identified as a potent protectant of neurons suffering from various adverse conditions. A wide array of literature indicates that endogenous or exogenous recombinant human erythropoietin and its variants activate cell signaling that initiates survival-promoting events in neurons and neuronal cells. This chapter gives an overview of the pro-survival signaling induced by endogenous and exogenous erythropoietin in vitro and in vivo and provides methods to further investigate the intracellular signaling. It is important to know that EPO is neuroprotective, but it will greatly enhance our chances to establish EPO as a new drug candidate if we know how EPO protects neurons.

The descriptions below summarize our current knowledge in non-neuronal and neuronal signaling pathways induced by EPO. The signaling pathways involved in EPO are multiple; some are well known whereas others are still under intense investigation and few are observed in very specific cell types. It is important to note that neuronal signaling events triggered by EPO are still incomplete and require further research. Therefore, excellent review articles that explore specific EPO-signaling events are referenced.

Key words

JAK2 Primary neuronal cultures Akt kinase assay Real time PCR Western blotting Immunoprecipitation 


  1. 1.
    Davis JM, Arakawa T, Strickland TW, Yphantis DA (1987) Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells. Biochemistry 26(9):2633–2638PubMedCrossRefGoogle Scholar
  2. 2.
    Yoon D, Ponka P, Prchal JT (2011) Hypoxia. 5. Hypoxia and hematopoiesis. Am J Physiol Cell Physiol 300(6):C1215–C1222. doi: 10.1152/ajpcell.00044.2011 PubMedCrossRefGoogle Scholar
  3. 3.
    Arcasoy MO (2008) The non-haematopoietic biological effects of erythropoietin. Br J Haematol 141(1):14–31. doi: 10.1111/j.1365-2141.2008.07014.x PubMedCrossRefGoogle Scholar
  4. 4.
    Juul SE, Stallings SA, Christensen RD (1999) Erythropoietin in the cerebrospinal fluid of neonates who sustained CNS injury. Pediatr Res 46(5):543–547PubMedCrossRefGoogle Scholar
  5. 5.
    Fu A, Hui EK, Lu JZ, Boado RJ, Pardridge WM (2010) Neuroprotection in stroke in the mouse with intravenous erythropoietin-Trojan horse fusion protein. Brain Res 1369:203–207. doi: S0006-8993(10)02440-6 (pii)  10.1016/j.brainres.2010.10.097 Google Scholar
  6. 6.
    Kapitsinou PP, Liu Q, Unger TL, Rha J, Davidoff O, Keith B, Epstein JA, Moores SL, Erickson-Miller CL, Haase VH (2010) Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia. Blood 116(16):3039–3048. doi: 10.1182/blood-2010-02-270322 PubMedCrossRefGoogle Scholar
  7. 7.
    Youssoufian H, Longmore G, Neumann D, Yoshimura A, Lodish HF (1993) Structure, function, and activation of the erythropoietin receptor. Blood 81(9):2223–2236PubMedGoogle Scholar
  8. 8.
    Watowich SS (2011) The erythropoietin receptor: molecular structure and hematopoietic signaling pathways. J Investig Med. doi: 10.231/JIM.0b013e31820fb28c
  9. 9.
    Jelkmann W (2004) Molecular biology of erythropoietin. Intern Med 43(8):649–659PubMedCrossRefGoogle Scholar
  10. 10.
    Ghezzi P, Bernaudin M, Bianchi R, Blomgren K, Brines M, Campana W, Cavaletti G, Cerami A, Chopp M, Coleman T, Digicaylioglu M, Ehrenreich H, Erbayraktar S, Erbayraktar Z, Gassmann M, Genc S, Gokmen N, Grasso G, Juul S, Lipton SA, Hand CC, Latini R, Lauria G, Leist M, Newton SS, Petit E, Probert L, Sfacteria A, Siren AL, Talan M, Thiemermann C, Westenbrink D, Yaqoob M, Zhu C (2010) Erythropoietin: not just about erythropoiesis. Lancet 375(9732):2142. doi: 10.1016/S0140-6736(10)60992-0 PubMedCrossRefGoogle Scholar
  11. 11.
    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(2):227–236PubMedCrossRefGoogle Scholar
  12. 12.
    Chateauvieux S, Grigorakaki C, Morceau F, Dicato M, Diederich M (2011) Erythropoietin, erythropoiesis and beyond. Biochem Pharmacol. doi: 10.1016/j.bcp.2011.06.045
  13. 13.
    Socolovsky M, Nam H, Fleming MD, Haase VH, Brugnara C, Lodish HF (2001) Ineffective erythropoiesis in Stat5a(−/−)5b(−/−) mice due to decreased survival of early erythroblasts. Blood 98(12):3261–3273PubMedCrossRefGoogle Scholar
  14. 14.
    Koury MJ, Bondurant MC (1991) The mechanism of erythropoietin action. Am J Kidney Dis 18(4 Suppl 1):20–23PubMedGoogle Scholar
  15. 15.
    Simon MC, Pevny L, Wiles MV, Keller G, Costantini F, Orkin SH (1992) Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells. Nat Genet 1(2):92–98. doi: 10.1038/ng0592-92 PubMedCrossRefGoogle Scholar
  16. 16.
    Lei H, Quelle FW (2009) FOXO transcription factors enforce cell cycle checkpoints and promote survival of hematopoietic cells after DNA damage. Mol Cancer Res 7(8):1294–1303. doi: 10.1158/1541-7786.MCR-08-0531 PubMedCrossRefGoogle Scholar
  17. 17.
    Kashii Y, Uchida M, Kirito K, Tanaka M, Nishijima K, Toshima M, Ando T, Koizumi K, Endoh T, Sawada K, Momoi M, Miura Y, Ozawa K, Komatsu N (2000) A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of ­phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction. Blood 96(3):941–949PubMedGoogle Scholar
  18. 18.
    Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281(5381):1312–1316PubMedCrossRefGoogle Scholar
  19. 19.
    Marti HH, Wenger RH, Rivas LA, Straumann U, Digicaylioglu M, Henn V, Yonekawa Y, Bauer C, Gassmann M (1996) Erythropoietin gene expression in human, monkey and murine brain. Eur J Neurosci 8(4):666–676PubMedCrossRefGoogle Scholar
  20. 20.
    Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, Petit E (1999) A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab 19(6):643–651. doi: 10.1097/00004647-199906000-00007 PubMedCrossRefGoogle Scholar
  21. 21.
    Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R (1994) A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J Biol Chem 269(30):19488–19493PubMedGoogle Scholar
  22. 22.
    Liu C, Shen K, Liu Z, Noguchi CT (1997) Regulated human erythropoietin receptor expression in mouse brain. J Biol Chem 272(51):32395–32400PubMedCrossRefGoogle Scholar
  23. 23.
    Tsai PT, Ohab JJ, Kertesz N, Groszer M, Matter C, Gao J, Liu X, Wu H, Carmichael ST (2006) A critical role of erythropoietin receptor in neurogenesis and post-stroke recovery. J Neurosci 26(4):1269–1274. doi: 10.1523/JNEUROSCI.4480-05.2006 PubMedCrossRefGoogle Scholar
  24. 24.
    Wu H, Lee SH, Gao J, Liu X, Iruela-Arispe ML (1999) Inactivation of erythropoietin leads to defects in cardiac morphogenesis. Development 126(16):3597–3605PubMedGoogle Scholar
  25. 25.
    Digicaylioglu M, Bichet S, Marti HH, Wenger RH, Rivas LA, Bauer C, Gassmann M (1995) Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci U S A 92(9):3717–3720PubMedCrossRefGoogle Scholar
  26. 26.
    Baciu I, Oprisiu C, Derevenco P, Vasile V, Muresan A, Hriscu M, Chis I (2000) The brain and other sites of erythropoietin production. Rom J Physiol 37(1–4):3–14PubMedGoogle Scholar
  27. 27.
    Siren AL, Knerlich F, Poser W, Gleiter CH, Bruck W, Ehrenreich H (2001) Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain. Acta Neuropathol 101(3):271–276PubMedGoogle Scholar
  28. 28.
    Sugawa M, Sakurai Y, Ishikawa-Ieda Y, Suzuki H, Asou H (2002) Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation. Neurosci Res 44(4):391–403PubMedCrossRefGoogle Scholar
  29. 29.
    Weidemann A, Kerdiles YM, Knaup KX, Rafie CA, Boutin AT, Stockmann C, Takeda N, Scadeng M, Shih AY, Haase VH, Simon MC, Kleinfeld D, Johnson RS (2009) The glial cell response is an essential component of hypoxia-induced erythropoiesis in mice. J Clin Invest 119(11):3373–3383. doi: 10.1172/JCI39378 PubMedGoogle Scholar
  30. 30.
    Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116(1):201–211PubMedGoogle Scholar
  31. 31.
    Hensey C, Gautier J (1998) Programmed cell death during Xenopus development: a spatio-temporal analysis. Dev Biol 203(1):36–48. doi: 10.1006/dbio.1998.9028 PubMedCrossRefGoogle Scholar
  32. 32.
    Digicaylioglu M, Lipton SA (2001) Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature 412(6847):641–647. doi: 10.1038/35088074, 35088074 (pii)PubMedCrossRefGoogle Scholar
  33. 33.
    Kaul M, Lipton SA (1999) Chemokines and activated macrophages in HIV gp120-induced neuronal apoptosis. Proc Natl Acad Sci U S A 96(14):8212–8216PubMedCrossRefGoogle Scholar
  34. 34.
    Budd SL, Lipton SA (1999) Signaling events in NMDA receptor-induced apoptosis in cerebrocortical cultures. Ann N Y Acad Sci 893:261–264PubMedCrossRefGoogle Scholar
  35. 35.
    Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci U S A 92(16):7162–7166PubMedCrossRefGoogle Scholar
  36. 36.
    D’Emilia DM, Lipton SA (1999) Ratio of S-nitrosohomocyst(e)ine to homocyst(e)ine or other thiols determines neurotoxicity in rat cerebrocortical cultures. Neurosci Lett 265(2):103–106PubMedCrossRefGoogle Scholar
  37. 37.
    Nicotera P, Ankarcrona M, Bonfoco E, Orrenius S, Lipton SA (1997) Neuronal necrosis and apoptosis: two distinct events induced by exposure to glutamate or oxidative stress. Adv Neurol 72:95–101PubMedGoogle Scholar
  38. 38.
    Garden GA, Budd SL, Tsai E, Hanson L, Kaul M, D’Emilia DM, Friedlander RM, Yuan J, Masliah E, Lipton SA (2002) Caspase cascades in human immunodeficiency virus-associated neurodegeneration. J Neurosci 22(10):4015–4024. doi: 20026351 PubMedGoogle Scholar
  39. 39.
    Sinclair AM, Coxon A, McCaffery I, Kaufman S, Paweletz K, Liu L, Busse L, Swift S, Elliott S, Begley CG (2010) Functional erythropoietin receptor is undetectable in endothelial, cardiac, neuronal, and renal cells. Blood 115(21):4264–4272. doi: 10.1182/blood-2009-10-248666 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Murat Digicaylioglu
    • 1
  1. 1.Departments of Neurosurgery and PhysiologyUniversity of Texas Health Science CenterSan AntonioUSA

Personalised recommendations