Inhibition of the erythropoietin-induced erythroid differentiation by granulocyte-macrophage colony-stimulating factor in the human UT-7 cell line is not due to a negative regulation of the erythropoietin receptor

Abstract

The human pluripotent UT-7 cell line is growth factor-dependent for proliferation and differentiation. We have previously shown that (1) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) induce a myeloid and erythroid pattern of differentiation, respectively; (2) GM-CSF acts predominantly over Epo for cell differentiation; (3) GM-CSF induces a rapid downmodulation (4 hours) of Epo receptors (Epo-R) at the mRNA and binding site levels; and (4) in contrast, Epo has no effect on GM-CSF receptor (GM-CSF-R) expression. These results suggested that UT-7 cell commitment or differentiation may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors. To test this hypothesis, we introduced and expressed the murine Epo-R (muEpo-R) in UT-7 cells using a retroviral strategy. Two retroviral vectors were constructed: one carrying the neomycin resistance gene, and another carrying a mouse Epo-R cDNA devoid of its regulatory untranslated 3′ sequence placed under the transcriptional control of the viral long terminal repeat element (LTR) and the neomycin resistance gene. Three UT-7/Epo-R infected clones (12, 6, 10) and one UT-7/neomycin clone (Neo) were selected in medium containing G418. After growth factor deprivation (18 hours), Epo-Rs were expressed at the same level (approximately 6,000 receptors per cell) in all four clones 12, 6, 10, Neo, and in parental UT-7 cells, and exhibited similar affinity (0.1 to 0.2 nmol/L). Cross-linking experiments showed that Epo is associated with three proteins of about 66, 85, and 100 kD in cells of parental UT-7, as well as in cells of clones 10 and 12. An inhibitory antibody directed specifically against the human Epo-R (huEpo-R Ab) abolished almost completely the cross-linking on parental UT-7 cells, but not on cells of clone 12, demonstrating that more than 90% cell surface Epo-Rs were of murine origin. The presence of GM-CSF significantly reduced the number of Epo-Rs expressed on parental UT-7 cells, but not on cells of clones 12, 10, and 6. HuEpo-R Ab inhibited Epo-induced parental UT-7 cell growth, but not that of cells of clone 12, suggesting that the muEpo-R is able to induce human UT-7 cell proliferation. When cells of clone 12 were switched from a medium containing GM-CSF to one with Epo, cell surface glycophorin A (GPA) was induced, as in parental UT-7 cells without inhibition by the huEpo-R Ab, demonstrating that the muEpo-R is also able to transduce a differentiation signal in human cells. However, in cells of clones 12, 6, 10 and Neo, as well as in parental UT-7 cells, the induction of GPA by Epo was inhibited by GM-CSF. This finding demonstrates that, although GM-CSF does not downregulate muEpo-R binding sites on UT- 7/muEpo-R infected clones, it still inhibits the effects of Epo on cell differentation. Therefore, hierarchical regulation induced by growth factors for cell commitment or differntiation more likely acts downstream of cell surface receptors at either the signal transduction or transcriptional levels.

Synergistic Effect of COX-2 Inhibitor on Paclitaxel-Induced Apoptosis in the Human Ovarian Cancer Cell Line OVCAR-3

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The combined effect of erythropoietin and granulocyte macrophage colony stimulating factor on liver regeneration after major hepatectomy in rats

“…A competition between EPO and GM-CSF has been reported in cells of the marrow [ 33 ]. In addition, it has been shown that GM-CSF can modulate EPO effects in certain leukemic cell line models of hematopoiesis, modulating events at the transcriptional and signal transduction level, or decreasing mRNA levels of EPO receptor [ 34 ]. Finally concentrations of hemopoietins have been found to play a key role in the final effect on cellular response [ 35 ].…”

Direct effect of erythropoietin on rat vascular smooth-muscle cell via a putative erythropoietin receptor

“…First, it is well known that even on erythroid precursor cells the number of EpoR expressed at the cell surface is extremely low [25]. Second, Epo receptors regularly circle from the cell surface to the cyto-plasm, depending on various factors such as the ambient Epo concentration [26,27]. In this regard it is possible that the Epo present in FCS binds to the cell EpoR, and therefore no available binding sites remain at the cell surface.…”