INHIBITION OF THE ERYTHROPOIETIN-INDUCED ERYTHROID-DIFFERENTIATION BYGRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR IN THE HUMAN UT-7 CELL-LINE IS NOT DUE TO A NEGATIVE REGULATION OF THE ERYTHROPOIETIN RECEPTOR

The human pluripotent UT-7 cell line is growth factor-dependent for pr oliferation and differentiation. We have previously shown that (1) gra nulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoie tin (Epo) induce a myeloid and erythroid pattern of differentiation, r espectively; (2) GM-CSF acts predominantly over Epo for cell different iation; (3) GM-CSF induces a rapid downmodulation (4 hours) of Epo rec eptors (Epo-R) at the mRNA and binding site levels; and (4) in contras t, Epo has no effect on GM-CSF receptor (GM-CSF-R) expression. These r esults suggested that UT-7 cell commitment or differentiation may be d irected by a hierarchical action of growth factors through an early an d rapid transmodulation of growth factor receptors. To test this hypot hesis, we introduced and expressed the murine Epo-R (muEpo-R) in UT-7 cells using a retroviral strategy. Two retroviral vectors were constru cted: one carrying the neomycin resistance gene, and another carrying a mouse Epo-R cDNA devoid of its regulatory untranslated 3' sequence p laced under the transcriptional control of the viral long terminal rep eat element (LTR) and the neomycin resistance gene. Three UT-7/Epo-R i nfected clones (12, 6, 10) and one UT-7/neomy-cin clone (Neo) were sel ected in medium containing G418. After growth factor deprivation (18 h ours), Epo-Rs were expressed at the same level (approximately 6,000 re ceptors per cell) in all four clones 12, 6, 10, Neo, and in parental U T-7 cells, and exhibited similar affinity (0.1 to 0.2 nmol/L). Cross-l inking experiments showed that Epo is associated with three proteins o f about 66, 85, and 100 kD in cells of parental UT-7, as well as in ce lls of clones 10 and 12. An inhibitory antibody directed specifically against the human Epo-R (huEpo-R Ab) abolished almost completely the c rosslinking on parental UT-7 cells, but not on cells of clone 12, demo nstrating that more than 90% cell surface Epo-Rs were of murine origin . The presence of GM-CSF significantly reduced the number of Epo-Rs ex pressed 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 no t that of cells of clone 12, suggesting that the muEpo-R is able to in duce human UT-7 cell proliferation. When cells of clone 12 were switch ed from a medium containing GM-CSF to one with Epo, cell surface glyco phorin A (GPA) was induced, as in parental UT-7 cells without inhibiti on by the huEpo-R Ab, demonstrating that the muEpo-R is also able to t ransduce 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 ind uction of GPA by Epo was inhibited by GM-CSF. This finding demonstrate s 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 differentiation. Therefore, hierarchical regulation induced by g rowth factors for cell commitment or differentiation more likely acts downstream of cell surface receptors at either the signal transduction or transcriptional levels. (C) 1996 by The American Society of Hemato logy.