Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In shortterm transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit + CD71 + erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit -CD71 + erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.

EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. Presently, the nature of EPOregulated survival genes has been investigated through transcriptome analyses of highly responsive, primary bone marrow erythroblasts. Two proapoptotic factors, Bim and FoxO3a, were rapidly repressed not only via the wild-type EPOR, but also by PY-deficient knocked-in EPOR alleles. In parallel, Pim1 and Pim3 kinases and Irs2 were induced. For this survival gene set, induction failed via a PY-null EPOR-HM allele, but was restored upon reconstitution of a PY343 STAT5-binding site within a related EPOR-H allele. Notably, EPOR-HM supports erythropoiesis at steady state but not during anemia, while EPOR-H exhibits near wildtype EPOR activities. EPOR-H and the wild-type EPOR (but not EPOR-HM) also markedly stimulated the expression of Trb3 pseudokinase, and intracellular serpin, Serpina-3G. For SERPINA-3G and TRB3, ectopic expression in EPOdependent progenitors furthermore significantly inhibited apoptosis due to cytokine withdrawal. BCL-XL and BCL2 also were studied, but in highly responsive Kit pos CD71 high Ter119 neg erythroblasts, neither was EPO modulated. EPOR survival circuits therefore include the repression of Bim plus IntroductionIn response to anemia, erythropoietin (EPO) is expressed by interstitial kidney and fetal liver cells via hypoxia-inducible transcription factor pathways. 1,2 As a secreted monomeric sialoglycoprotein, EPO then targets developing erythroblasts, and is essential for red cell formation during definitive bone marrow and fetal liver erythropoiesis. [3][4][5][6][7][8] Prospective roles for EPO in promoting primitive red cell formation in yolk sac also have recently been described. 9 Beyond this, recombinant EPO has been demonstrated in ischemia and other cell damage models to provide cytoprotective effects for injured renal, cardiac, retinal, and neuronal tissues. 10,11 Taken together, these considerations have heightened interest in the specific nature of key EPO action mechanisms, especially those associated with progenitor cell survival.EPO's prime effects are mediated via interactions with its dimeric single-transmembrane receptor (EPOR). [3][4][5][6][7][8]12 These interactions appear to evoke EPOR conformational events, 13 which are relayed to an upstream Janus kinase, JAK2 14 (and JAK2 likewise may preassemble with EPOR dimers at a juxtamembrane box1 domain). 15,16 JAK2, as activated via a Y1007 phosphorylation loop, 17 next stimulates 2 separable signal transduction pathways. First, JAK2 interestingly can support steady-state erythropoiesis via EPOR-PY-independent routes that, in part, may involve MEK1,2 and ERK1,2 stimulation. 18 Second, JAK2 also mediates the phosphorylation of 8 conserved EPOR cytoplasmic PY sites, which can then form a scaffold for the binding of up to 20 SH2-or PTB-domain encoding signal transduction factors and molecular adaptors. [6][7][8][19][20][21] Among conserve...

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...