Hemogloblin Switching in Sheep and Goats: Erythropoietin-Dependent Synthesis of Hemoglobin C in Goat Bone-Marrow Cultures

Abstract

The anemia-induced switch from hemo. In eukaryotes, the molecular mechanisms involved in gene activation and in the subsequent production of specific cell proteins are largely unknown. To study gene activation in mammals, a cell population is needed that can be stimulated to transcribe the messenger RNA for a particular protein in response to a defined stimulus. We have studied the erythropoietin-dependent synthesis of hemoglobin C (HbC) in goat (and sheep) bone-marrow cultures for this purpose. Erythropoietin, a glycoprotein hormone, appears to stimulate precursor blood cells in the bone marrow to differentiate into cells that produce hemoglobin (1-3). The type of hemoglobin produced by these cells is determined by the genetic makeup of the animal and by its stage of development. For example, sheep and goats homozygous for hemoglobin A (HbA) pass through a series of developmental stages during which they produce HbF (a2Y2), then HbC (aZ2c), andfinally-the adult HbA (aZ82A) (4-9). However, in response to severe anemia, an adult goat making HbA completely switches from the synthesis of HbA to HbC (4). There is a lag of 3 days between the induction of anemia and the detection of Oc globin synthesis in the marrow (10), suggesting that flc globin is produced by a newly stimulated population of precursor cells. As the anemia is relieved, HbA again is synthesized. The switch from MA to tic synthesis can be elicited in normal, nonanemic animals by injection of partially purified erythropoietin preparations (10-13).We have shown (14) that sheep and goats that are synthesizing HbA have only the mRNA for MA (and a) globin in their erythroid cells, while animals synthesizing HbC have only the mRNA for flc (and a) METHODSSource and Preparation of Cells. Goats used in this study were over 6 months old and had only HbA detectable in their peripheral blood, as determined by thin-layer electrophoresis on cellulose acetate (14). The animals were anesthesized by intravenous injection of sodium pentobarbital (60 mg/ml, 0.5 ml/kg) and exsanguinated through a femoral artery. The femurs and humeri were removed, their proximal and distal ends were cut off to expose the marrow cavity, and the marrow was extruded by flushing medium (Eagle's minimum essential medium for suspension culture; Microbiological Associates, Bethesda, Md.) through the bones. The cells were separated by being expelled from a 50-ml syringe through a. 20-gauge needle, were washed once with Eagle's medium, and then were resuspended in NCTC-109 medium (Microbiological Assoc.) and disaggregated by expulsion through 'successively smaller-bore syringe needles down to 25-gauge. The concentration of nucleated cells was adjusted to 0.5 to 1 X 101 cells.per ml by the addition of appropriate volumes of a culture medium (16) containing 5 ml of fetal-bovine serum, 2.5 ml of 7.5% NaHCO3, 1 ml of 200 mM iglutamine, 2 ml of penicillinstreptomycin (5000 units of each per ml), 86.7 ml of NCTC-109 medium (all of the preceding components were obtained from Microbiological ...

Effect of Hypoxia on Erythroblasts from Avian Fetal Liver: Adenosine Triphosphate Levels and Hemoglobin Synthesis

“…These two hemoglobins are synthesized in the same cells and the control of this transition appears to be at the nuclear level, probably via transcription (3,18,33). This is in contrast to the mechanism for control of differentiation in species which shift from a primitive to a definitive cell line.…”

βMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in β thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles

“…Consistently, accumulation of ␤ C globin mRNA in response to intense Epo stimulation was previously described in anemic sheep 5 and in sheep bone marrow cultures. 17 Drugs that reactivate fetal erythropoiesis in human erythroid cell cultures, such as hydroxyurea, 5-azacytydine and butyrate, increase ␥-globin mRNA amounts. 16 In contrast, it has been shown that the compensatory increase of ␤min-globin chain synthesis in untreated ␤ thalassemic mice affects mRNA translation rather than accumulation.…”

“…16 Increased ␤ C -globin mRNA amounts were also observed in anemic sheep 5 or in sheep bone marrow cultures stimulated with high doses of Epo. 17 It has been proposed that a more efficient translation of ␤min-globin mRNA than ␣-globin mRNA would account, at least partly, for the compensatory increase of ␤min-globin synthesis observed in ␤ thalassemic mice, in which serum Epo concentration is elevated. 18 However, how Epo improves the phenotype of ␤ thalassemic erythrocytes has not been directly investigated so far.…”

Hemoglobin switching in sheep and goats. VI. Commitment of erythroid colony-forming cells to the synthesis of betaC globin.

“…After an additional 24 h of incubation, the colonies were retrieved by trypsinization of the clots from 48 to 60 wells, washed, and lysed, and their products were analyzed by carboxymeth-ylcelluiose chromatography in 8 M urea phosphate buffer (3,16). To determine the pattern of globin synthesis in differentiating erythroid cells, bone marrow was placed directly in suspension culture and labeled between 0 and 11 h in vitro by addition of 300 /xCi of [aH]teucine (4). The cells were recovered and prepared for chromatography as previously described (16).…”

“…As the animal matures, hemoglobin C synthesis ceases and is replaced by the adult hemoglobin; hemoglobin A (c~d32 A) in sheep and hemoglobin A, D, or E (containing flA, /3D, and /3 ~, respectively) in goats. In bone marrow cells of adult animals, hemoglobin C production can be reinitiated in vivo by anemic stress or injection of ESF (7,10,23) and in vitro by exposure to ESF (1,3,4).…”