Globin Messenger RNA Activity in Erythroid Precursor Cells and the Effect of Erythropoietin

Abstract

Cell populations enriched for erythroid precursor cells were fractionated from 13-day fetal-mouse livers by a method of immune hemolysis. These preparations of precursors, contaminated by less than 7% hemoglobinized erythroblasts, synthesize globin at a rate less than 6% that of the unfractionated liver erythroid cell population. RNA was isolated from these precursor cells and assayed for globin mRNA activity in a cell-free system from Krebs ascites tumor. The 6-16S RNA fraction from precursor cells has less than 5% of the globin mRNA activity of RNA isolated from unfractionated populations. Precursor cells incubated with erythropoietin show an increment in the rate of synthesis of globin only after 5-10 hr of incubation. After 10 hr of culture with this hormone, precursor cells show a 6-to 10-fold increase in globin mRNA activity. These results suggest that the precursor cells of hepatic erythropoiesis, responsive to erythropoietin, do not contain globin mRNA in a biologically active form. Erythropoietin-induced differentiation of these cells to erythroblasts is associated with an increase in globin mRNA.Fetal-mouse liver erythropoiesis provides a model for study of mammalian cell differentiation (1). The erythroid cell population obtained from 13-day fetal livers contains cells at all stages of differentiation, from the immature, erythropoietinresponsive precursor to the fully hemoglobinized erythrocyte. Erythroid precursor cells can be concentrated from the livers of fetal mice of 12-and 13-days gestation (2). Incubation of these precursor cells with erythropoietin stimulates both proliferation and differentiation to cells that actively synthesize hemoglobin. The questions posed in the present study are: (i) Does the precursor cell population contain biologically active mRNA for globin and synthesize globin, and (ii) Does erythropoietin-induced stimulation of erythroid cell differentiation involve an increase in the amount of biologically active globin mRNA? The evidence indicates that precursor cell populations (contaminated with 2-7% hemoglobinized erythroblasts) contain less than 5% of the globin mRNA activity recovered from an unfractionated erythroid cell population from fetal liver, and that they synthesize globin at a rate less than 5% that of the unfractionated cells.Erythropoietin-induced differentiation of precursor cells is characterized by a marked increase in globin mRNA activity and in the rate of globin synthesis. (pH 7.4) and centrifuged at 750 X g for 3 min at 4°. The supernatant was removed, the nuclear fraction was washed once with 0.5 ml of this buffer, and the wash was combined with the first supernatant solution. (b) Mechanical lysis: the cells were suspended in 2 ml of 30 mM Tris-HCl-5 mM Mg acetate-10 mM KCl (pH 7.5) and lysed in a Dounce homogenizer with five strokes of the lightly-fitted pestle (5).

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

“…Since in fetal liver in vivo the time for transition of a basophilic erythroblast to a polychromatic erythroblast is a few hours (15), this implies that there may be some translational control of globin synthesis at this stage of fetal liver development. Using a sensitive assay, Terada et al (3) have shown that an immature erythroid cell preparation (obtained by treatment of 13.5-day fetal liver cells with antiserum against mature red blood cells and containing not more than 7% hemoglobinized erythroblasts) synthesizes a-and B-globin chains at less than 6% of the rate for total fetal liver cells. The same workers showed that the proportion of translatable globin mRNA in the 6 16S RNA fraction from these immature cells (determined by the ability to direct a-and B-globin synthesis in the Krebs ascites cell-free system) was less than 5% of the corresponding value for total fetal liver cells.…”

“…This work also demonstrated the presence of globin m R N A in basophiiic erythroblasts from 13-day fetal livers; whereas little globin m R N A could be detected in the more immature proerythroblasts. However, Terada et al (3) have not detected significant amounts of translatable globin m R N A in immature erythroid cells (a mixture of proerythroblasts and basophilic erythroblasts obtained by treatment of total fetal liver cells with an antiserum raised against mature red blood cells), as judged by the ability to direct globin synthesis in the Krebs ascites lysate system. These reports therefore suggest the interesting possibility that during erythroid cell differentiation globin m R N A may be synthesized before the immature erythroid cell has the capacity to translate it into globin chains.…”

Testosterone stimulation of a rapidly labeled, low-molecular-weight RNA fraction in human hepatic erythroid cells in culture.

“…One of the earliest actions of erythropoietin on its target cells is the stimulation of RNA synthesis (1)(2)(3)(4)(5)(6). We have found that primary cultures of erythroid cells from human fetal liver exhibited an enhanced synthesis of heme after addition of erythropoietin or testosterone and that the heme fraction associated with hemoglobin showed the highest stimulation (7).…”

Erythropoietic Differentiation in Colonies of Cells Transformed by Friend Virus

“…This effect of erythropoietin involves an early increase in the rate of synthesis of ribosomal RNA, followed by the accumulation of mRNA for globin, stimulation of globin synthesis, and appearance of erythrocyte membrane antigens (1)(2)(3). These biochemical events are associated with morphological changes characterized by development from an early proerythroblast to a nonnucleated reticulocyte.…”