Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7.
Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma-globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3).(ABSTRACT TRUNCATED AT 400 WORDS)
Nicolis
S
S
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Italy.
Ottolenghi
S
S
Papayannopoulou
T
T
Baiocchi
M
M
Migliaccio
G
G
Adamson
J
J
Migliaccio
A R
AR
D015152:Blotting, Northern; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D004921:Erythropoietin / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D050982:GATA1 Transcription Factor; D015870:Gene Expression; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism / Q000494:pharmacology; D017467:Receptors, Erythropoietin / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D007377:Interleukin-3; D017467:Receptors, Erythropoietin; D014157:Transcription Factors; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005914:Globins