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

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.

Fig. 1

High dose of EPO fails to support cell proliferation in EPO dependent Ep-FDC-P2. a EPO dependent growth curve. closed circle EPO only; open circle EPO plus anti EPO antibody R2 (1 mg mL⁻¹), b EPO dependent growth curve. closed circle EPO only; open triangle EPO plus anti EPO antibody R2 (1 mg mL⁻¹); closed triangle EPO plus anti EPO antibody R6 (1 mg mL⁻¹); open square EPO plus unrelated antibody CX-C (1 mg mL⁻¹). c purification of sEPOR from culture supernatant. lane 1 culture supernatant, lane 2 purified sEPOR by EPO immobilized to a column. +CHO shows N-linked glycosylation of sEPOR; -CHO shows lack of glycosylation of sEPOR. d EPO dependent growth curve. closed circle EPO only; closed triangle EPO plus 1 mg mL⁻¹ sEPOR; open triangle EPO plus 3 mg mL⁻¹ sEPOR. Each point is the mean ± S.D. of triplicate assay (a, b and d)

Fig. 2

EPO dependent growth curve. A high dose of EPO does not support murine and human EPO-dependent cell lines. a BaF/ER; b UT-7; c TF-1; cells were cultured with EPO and with or without sEPOR, respectively. closed circle EPO only; closed triangle EPO plus 1 mg mL⁻¹ sEPOR; open triangle EPO plus 3 mg mL⁻¹ sEPOR. Each point is the mean ± S.D. of triplicate assay

Fig. 3

High dose of EPO indiced electrophoresis DNA fragmentation. Chromosomal DNA was prepared at times as indicated and separated by agarose gel. a time course of DNA fragmentation. + cells were cultured with 10 U mL⁻¹ EPO or 100 μg mL⁻¹ R2, ++ with 1 × 10⁴ U mL⁻¹ EPO, b DNA fragmentation analysis. Cells were cultured with or without EPO. + 10 U mL⁻¹ EPO or 1.5 mg mL⁻¹ sEPOR; ++ 1 × 10⁴ U mL⁻¹ EPO

Fig. 4

IL-3 supports cell proliferation in the presence of high dose of EPO. a IL-3 dependent growth curve at high EPO concentration. closed circle EPO only; closed square EPO plus murine IL-3 (100 U mL⁻¹), each point is the mean ± S.D. of triplicate assay. b time course of DNA fragmentation. + cells were cultured with 10 U mL⁻¹ EPO or 100 U mL⁻¹ IL-3, ++ with 1 × 10⁴ U mL⁻¹ EPO

Fig. 5

A high dose of EPO fails to activate the phosphorylation of EPOR and JAK2 kinase. a phosphorylation of EPOR by EPO. b high dose of EPO inhibits the phosphorylation of EPOR. c phosphorylation of p130. d high dose of EPO fails to activate the phosphorylation of p130. e p130 is JAK2. + Ep-FDC-P2 cells were cultured with 10 U mL⁻¹; ++ with 1 × 10⁴ U mL⁻¹ EPO