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EPO modulation of cell-cycle regulatory genes, and cell division, in primary bone marrow erythroblasts.

ABSTRACT: Erythropoietin (EPO's) actions on erythroblasts are ascribed largely to survival effects. Certain studies, however, point to EPO-regulated proliferation. To investigate this problem in a primary system, Kit(pos)CD71(high) erythroblasts were prepared from murine bone marrow, and were first used in the array-based discovery of EPO-modulated cell-cycle regulators. Five cell-cycle progression factors were rapidly up-modulated: nuclear protein 1 (Nupr1), G1 to S phase transition 1 (Gspt1), early growth response 1 (Egr1), Ngfi-A binding protein 2 (Nab2), and cyclin D2. In contrast, inhibitory cyclin G2, p27/Cdkn1b, and B-cell leukemia/lymphoma 6 (Bcl6) were sharply down-modulated. For CYCLIN G2, ectopic expression also proved to selectively attenuate EPO-dependent UT7epo cell-cycle progression at S-phase. As analyzed in primary erythroblasts expressing minimal EPO receptor alleles, EPO repression of cyclin G2 and Bcl6, and induction of cyclin D2, were determined to depend on PY343 (and Stat5) signals. Furthermore, erythroblasts expressing a on PY-null EPOR-HM allele were abnormally distributed in G0/G1. During differentiation divisions, EPOR-HM Ter119(pos) erythroblasts conversely accumulated in S-phase and faltered in an apparent EPO-directed transition to G0/G1. EPO/EPOR signals therefore control the expression of select cell-cycle regulatory genes that are proposed to modulate stage-specific decisions for erythroblast cell-cycle progression.

SUBMITTER: Fang J

PROVIDER: S-EPMC1988929 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.

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EPO modulation of cell-cycle regulatory genes, and cell division, in primary bone marrow erythroblasts.

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