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 downmodulated. For CYCLIN G2, ectopic expression also proved to selectively attenuate EPO-dependent UT7epo cellcycle 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 ( IntroductionThrough gene disruption experiments, 1 erythropoietin (EPO) and its dimeric single transmembrane receptor (EPOR) are known to be essential for erythroblast formation beyond a colony-forming unit-erythroid stage. In these erythroid progenitor cells, the EPOR plays a key survival role 2,3 and couples EPO to several antiapoptotic pathways. These include EPOR PY479 coupling to p85alpha PI3-kinase, thymoma viral proto-oncogene 1 (AKT), mammalian target of rapamycin (mTOR), and forkhead box O3a (FOXO3A) signal transduction factors. [4][5][6] A constitutively active AKT mutant also can rescue Janus kinase 2 (JAK2)-deficient fetal liver erythroblast development, 7 and ectopically expressed Bcl-2-like-1 (BCL-XL) can support erythroblast formation in the absence of Stat5 and/or EPO signaling. 8 In addition, an EPOR PY343 Stat5 binding site may promote Bcl-xl gene expression, 9,10 and recently this cytoplasmic phosphotyrosine site (and associated transduction pathways) have been shown to support stress erythropoiesis. 11 Erythroid progenitor cells are also sharply regulated within the contexts of both renewal and differentiation divisions. 12 Differentiation divisions occur during late-stage development, and exhibit a shortened G1-phase. 13 In such differentiating erythroblasts, E2F transcription factor 4 (E2F4) interestingly has been shown to selectively promote proliferation, and to possibly regulate several cell-cycle-associated genes (eg, Cyclin A2 [Ccna2], minichromosome maintenance deficient 2 mitotin (Mcm2) and proviral insertion Emi 1 [Emi1]). 14,15 E2F4-deficient peripheral erythrocytes also are macrocytic, implicating E2F4 effects on size control. Renewal erythroid divisions, in contrast, exhibit more standard cell-cycle properties, and are stimulated during hypoxic stress and anemia. 12 The nature of factors that exert primary control over erythroid progenitor renewal divisions, however, is less clear.Several previous studies (predominantly in cell line models) have suggested that EPO also may modulate erythroid progenitor proli...

Epo's erythropoietic capacity is ascribed largely to its antiapoptotic actions. In part via gene profiling of bone marrow erythroblasts, Epo is now shown to selectively down-modulate the adhesion/migration factors chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4) and to up-modulate growth differentiation factor-3 (Gdf3), oncostatin-M (OncoM), and podocalyxin like-1 (PODXL). For PODXL, Epo dose-dependent expression of this CD34-related sialomucin was discovered in Kit(+)CD71(high) proerythroblasts and was sustained at subsequent Kit(-)CD71(high) and Ter119(+) stages. In vivo, Epo markedly induced PODXL expression in these progenitors and in marrow-resident reticulocytes. This was further associated with a rapid release of PODXL(+) reticulocytes to blood. As studied in erythroblasts expressing minimal Epo receptor (EpoR) alleles, efficient PODXL induction proved dependence on an EpoR-PY343 Stat5 binding site. Moreover, in mice expressing an EpoR-HM F343 allele, compromised Epo-induced PODXL expression correlated with abnormal anucleated red cell representation in marrow. By modulating this select set of cell-surface adhesion molecules and chemokines, Epo is proposed to mobilize erythroblasts from a hypothesized stromal niche and possibly promote reticulocyte egress to blood.

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