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

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Abstract

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.

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Abbreviations

EPO:

Erythropoietin

EPOR:

EPO receptor

sEPOR:

Soluble form of EPOR

CFU-E:

Colony-forming unit erythroid

IL-3:

Interleukin-3

MEM:

Minimum essential medium

FCS:

Fetal calf serum

MTX:

Methotrexate

MTT:

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

JAK:

Janus family of nonreceptor-type protein tyrosine kinase

STAT:

Signal transducers and activators of transcription

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto, 606-8502, Japan

    Satoshi Abe, Ryuzo Sasaki & Seiji Masuda

  2. Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan

    Ryuzo Sasaki & Seiji Masuda

Authors
  1. Satoshi Abe
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  2. Ryuzo Sasaki
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  3. Seiji Masuda
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Correspondence to Seiji Masuda.

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Abe, S., Sasaki, R. & Masuda, S. An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines. Cytotechnology 63, 101–109 (2011). https://doi.org/10.1007/s10616-011-9345-x

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  • DOI: https://doi.org/10.1007/s10616-011-9345-x

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