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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
O Hermine, A Dubart, F Porteux, P Mayeux, M Titeux, D Dumenil and W Vainchenker
INSERM U 362, Institut Gustave Roussy, Villejuif, France.
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.
Volume 87,
Issue 5,
pp. 1746-1753,
03/01/1996
Copyright © 1996 by The American Society of Hematology
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