Survival Of Proerythroblasts Research Articles

Eltrombopag Improves Erythroid Differentiation in a Human iPSC Model of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow (BM) failure syndrome primarily characterized by defective erythropoiesis. In most patients, pathogenic heterozygous mutations have been identified in genes encoding ribosomal proteins (RP). The resulting RP haploinsufficiency was recently shown to delay globin protein translation in erythroid cells, whereas synthesis of heme, the nonprotein iron-containing component of hemoglobin, proceeds normally (Yang et al., Sci Transl Med 2016). Because heme is first synthesized at or just before the proerythroblast stage, free heme is in excess of globin in these cells. High levels of free heme induce proerythroblast cell death, and erythroid differentiation thus halts at the earlier BFU-E/CFU-E progenitor stage. Consistent with these observations, inhibition of heme synthesis with succinylacetone was previously shown to improve erythroid differentiation of DBA marrow cells in vitro. In this study, we investigated whether eltrombopag (Epag), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BM failure (Olnes et al., NEJM 2012; Townsley et al., NEJM 2017), could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced reactive oxygen species (ROS) and increased proerythroblast survival and maturation. To test this possibility,we first established an induced pluripotent stem cell (iPSC) model of DBA by reprogramming mononuclear cells (MNCs) from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line.RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTMmonolayer-based approach (Ruiz et al., BioRxiv 2019). Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+primitive erythroid progenitors (P1), CD71+CD45loEPOR-proerythroblasts (P2), and more mature CD71+CD45-EPOR-erythroblasts (P3) (Figure). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage (Figure). Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitor's inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 µg/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations (Figure). Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA. Figure Disclosures No relevant conflicts of interest to declare.

Adenosine Signaling-Mediated Metabolic Reprogramming Regulates Erythropoiesis

Erythropoiesis is an extremely dynamic process finely regulated by cytokines, hormones, and growth factors at transcriptional and translational levels. Stress-induced erythropoiesis is defined as a stimulated basal erythropoiesis with expansion of the erythroid progenitor pool, associated with reticulocytosis and splenomegaly. Stress erythropoiesis is stimulated under the condition of insufficient oxygen availability such as high altitude, blood loss, infection, and anemia. Thus, stress erythropoiesis is an important stress adaptive response for survival. Although stress erythropoiesis has been long speculated to be linked with increased metabolic requirements, until recent two years with innovative metabolomics profiling and state of art isotopically labelled metabolic flux approaches, the filed has evolved and revealed that enhanced glucose and glutamine metabolism is essential for stress erythropoiesis. However, molecular basis underlying metabolic reprogramming to enhance glucose metabolism and subsequently stress erythropoiesis remains unclear.To address this question, we conducted both human and mouse studies. First, we found that plasma adenosine is rapidly induced and associated with stress erythropoiesis features including increased hematocrit (HCT), hemoglobin (Hb) mass and reticulocytes in healthy human volunteers at high altitude and in mice exposed to hypoxia mimicking high altitude. Follow-up mouse genetic studies showed that activation of adenosine signaling via erythroid ADORA2B promotes the survival and expansion of proerythroblasts both in spleen and bone marrow and in this way contributes to hypoxia-induced stress erythropoiesis independent of erythropoietin. Using unbiased high-throughput metabolic profiling, we identified that erythroid ADORA2B contributes to an overall hypoxia metabolic reprogramming with substantial increased glycolysis in proerythroblast progenitors in mice. Finally, using primary human CD34+ hematopoietic stem cells culture, we showed that adenosine analogue and ADORA2B agonist promote the survival and expansion of erythroid progenitors in a time and dose-dependent manner. Taken together, both human and mouse studies identify that adenosine ADORA2B is a previously unrecognized purinergic signaling underlying hypoxia-induced erythropoiesis by facilitating expansion and survival of proerythroblasts, and highlight that enhancing this pathway is a potential strategy to induce erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Defining an EPOR- Regulated Transcriptome for Primary Progenitors, including Tnfr-sf13c as a Novel Mediator of EPO- Dependent Erythroblast Formation

Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.

FLVCR Deletion Does Not Impact Normal Hematopoietic Stem Cell Function but Is Necessary for Erythroid and T-Cell Maturation and Enhances Platelet Formation

Abstract 2363Hematopoietic stem cells (HSCs) are mainly in a quiescent state, but activate and proliferate in response to stress. To maintain quiescence, as well as respond to activation signals, HSCs must coordinately regulate cell cycle status versus metabolic demand. Mismatch in these pathways can lead to HSC exhaustion, failure or malignant transformation. One metabolic pathway that is essential to all cells is heme biosynthesis. Heme can act directly as a signaling molecule by modulating the function of transcriptional (Bach1) or translational (eIFa2 kinase) regulators. In addition, heme can act indirectly through the action of hemoproteins (hemoglobin, oxidases, peroxidases, cytochromes). However, excess free intracellular heme is toxic, therefore organisms have developed “safety valve” pathways to regulate intracellular heme concentrations. The most well studied mechanism is free heme degradation by the heme-oxygenase pathway. We have recently described a second pathway to prevent intracellular heme excess, namely heme export by the cytoplasmic transporter FLVCR. Previously we have shown that FLVCR is essential for erythropoiesis, as FLVCR−/− mice die of anemia (Keel et. al., Science 319: 825, 2008). FLVCR is expressed in many other tissues and blood cells, including HSCs (Lucas et. al., Blood 106:51, 2005). This suggests that FLVCR may be important in HSC function or the development of other lineages, besides red cells. We used a competitive transplantation model to determine the effect of FLVCR deletion on HSC function and lineage maturation. FLVCR−/− or wild type (WT) donor cells and equal numbers of competitor cells were transplanted into lethally irradiated, syngeneic recipients. FLVCR−/− marrow cells reconstitute the granulocyte compartment equally as well as WT cells in serial transplantation (up to tertiary transplants). Equal contributions also remain after myelotoxic stress (radiation or arsenic trioxide). Therefore, FLVCR may be dispensable for normal HSC function under steady-state and stress conditions. As anticipated, FLVCR is required for proerythroblast survival and maturation. Eight weeks after competitive transplantation with FLVCR−/− donor cells, 100% of red cells are derived from competitor cells. This is also seen when the ratio of FLVCR−/−:competitor cells is increased to 95:5 from 50:50. FLVCR−/− cells fail to mature past the proerythroblast stage (CD71pos lin-Ter119neg or lo) and undergo significantly more apoptosis than WT donor cells (88% vs. 36%, p<0.001). FLVCR deletion also blocks T-cell development. Due to biological variability in our experimental model, T-cell numbers were normalized to granulocytes in each determination, in each mouse and expressed as a percentage of the expected value. At 5 weeks post-transplant, the percentage of FLVCR−/− derived T-cells was 65.8% of the percentage of FLVCR−/− derived granulocytes. The percentage of WT derived T-cells was 93.4% of the percentage of WT derived granulocytes (p<0.02). Over time, FLVCR−/− derived T-cells steadily decreased, dropping to 22% of expected values by week 24, and remaining low, whereas T-cells derived from control, WT donor cells remained stable (74–98% of expected values). FLVCR−/− T-cells are arrested at the double positive (CD4+CD8+) stage (Philip et. al., Blood (ASH Abstracts) 114:913, 2009). Surprisingly, loss of FLVCR improves platelet production. FLVCR−/− donor cells make up ∼60% of all platelets (avg. 1.71 x expected, when normalized to granulocytes). In the separate control cohort competitively transplanted with WT donor cells, ∼40% of the platelet population was derived from WT cells (avg. 0.91 x expected, when normalized to granulocytes). Our studies suggest that the increased platelet production likely results from increased megakaryocyte ploidy (e.g. avg. 14.2N in FLVCR−/− vs 5.2N in control) and not from increased megakaryocyte progenitors. Of note, FLVCR−/− deleted mice have thrombocytosis (2221/μL vs 1008/μL, p<0.001 in littermate controls), a finding we initially dismissed as a stress response to severe anemia. Together, these findings reveal stage specific roles for FLVCR in discrete hematopoietic lineages and support the broader hypothesis that heme excess might impair cell division, leading to decreased red and T-cell production, but improved endomitosis and increased platelet production. Disclosures:No relevant conflicts of interest to declare.

Why Do Erythroid Cells Need to Export Heme?

Abstract SCI-24Red cells are anucleated sacs involved in gas exchange and most importantly oxygen delivery. Over 90% of their protein content is hemoglobin. We inadvertently determined that the feline leukemia virus C receptor (FLVCR), a cytoplasmic membrane transporter that is required for proerythroblast survival, exports heme (1). Mice in which Flvcr is ablated die in utero from erythroid failure and adult mice in which Flvcr is deleted develop a severe macrocytic anemia (2). These observations led to an important question that will be addressed in this session: Why should developing erythroid cells need to export heme when its adequate supply in mature red cells is so critical? Although FLVCR is ubiquitously expressed in different human tissues by northern analyses and RT-PCR, western blot data suggest it is most abundant in liver, duodenum, brain, placenta, spleen, marrow, and uterus, which are sites of high heme synthesis or flux, and thus are cells that might need protection from heme toxicities. FLVCR's impact on erythropoiesis is cell-autonomous – normal mice transplanted with Flvcr-deleted marrow develop severe macrocytic anemia and Flvcr-deleted mice transplanted with wild-type (WT) marrow cells regain normal erythropoiesis. Erythropoiesis is also rescued when mice are transplanted with Flvcr-deleted marrow transduced with a retroviral vector encoding huFLVCR. These observations led to the hypothesis that at the time during erythroid differentiation when heme synthesis initiates and before globin synthesis (which is transcriptionally (via Bach1) and translationally (via HRI regulated by heme) is significantly robust, cells must export heme so that heme synthesis does not exceed use. Erythroid cells may be especially vulnerable to heme toxicity as ALA-S2, the first and rate-limiting step of heme synthesis (in contrast to ALA-S1, the isoform of nonerythroid cells) is not inhibited by heme. Importantly, although FLVCR clearly exports heme from cells, we still do not have definitive proof that failure to export heme from proerythroblasts causes macrocytic anemia. Genetic studies to directly test this are under way and results will be presented. Data from competitive reconstitution studies with Flvcr-deleted and WT marrow will also be presented. When the cells are transplanted into irradiated WT mice at ratios from 50:50 to 95:5, the recipient's red cells, as expected, are exclusively WT in phenotype. Few Ter119 + Flvcr-deleted cells are seen and CD71+ Ter119 low (proerythroblasts) have increased apoptosis. Of note, the T cell differentiation of Flvcr-deleted cells is also blocked at a specific stage (CD4, CD8 double positive cells in thymus) and interestingly, platelet production is increased. In preliminary studies, it appears that the enhanced platelet production occurs at the level of megakaryocytes (increased ploidy) and does not result from biased decision-making of a common BFU-meg progenitor. Given the emerging links between metabolism and cell cycle and recognizing that heme synthesis begins with succinyl CoA, a Krebs cycle intermediate, it seems likely that understanding FLVCR biology and heme trafficking will provide novel insights into these lineage and stage-specific decisions. Disclosures:No relevant conflicts of interest to declare.

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

Evidence That Heme Export Via the Feline Leukemia Virus, Subgroup, C Receptor (FLVCR) Is Carrier Dependent and Its Physiological Implications.

FLVCR is a heme export protein that is required for proerythroblast survival (Cell 18:757, 2004), macrophage heme-iron recycling, and systemic iron homeostasis (ASH abstract, 2007). However, the mechanism and regulation of export through this and related major facilitator superfamily (MFS) members (e.g., Glut4, GlpT) is unknown. To gain insights into the structure and function of FLVCR, we studied the export of zinc mesoporphyrin (ZnMP), a fluorescent heme analog that traffics like heme and blocks heme oxygenase (HO) activity. NRK cells engineered to express huFLVCR were loaded with ZnMP for 30 min, then incubated at 37°C × 90 min with no carrier protein, albumin, or hemopexin (HPXN). In the absence of carrier protein, no export was seen. Export was near complete in the presence of 152 μM (1%) albumin, but less extensive with 15.2 μM albumin and absent with 1.52 μM albumin. Interestingly, HPXN appeared 100 fold more efficacious in inducing ZnMP export, as the residual ZnMP fluorescence in the presence of 0.152 μM HPXN was equivalent to residual intensity in the presence of 15.2 μM albumin. Comparable results were obtained in studies of human blood macrophages. Heme trafficking was next quantitated using 55Fe-heme and a similar study design (ZnMP was added to block HO activity). In the presence of 1.52 μM albumin, 6% of the 55Fe-heme load was exported, while 52% was exported in the presence of 1.52 μM HPXN. When the albumin concentration was increased 100 fold to 152 μM, 67% was exported. As Kds are ∼10 nM and <1 pM, for albumin and HPXN, respectively, our data argue that the rate of heme export depends on the avidity of this binding. Carrier proteins coordinate the heme-iron through two histidines and stabilize this interaction with multiple aromatic amino acids lining the heme-binding pocket. Of note, there are three conserved histidine residues in FLVCR, two of which (aa 145 and 198) are on adjacent extracellular loops, and based upon known MFS family member structures (Protein Sci. 13:1832, 2004), are potentially optimally located to coordinate the heme-iron exiting the channel. Adjacent transmembrane regions contain several aromatic residues capable of lining a heme pocket. These histidine residues are conserved in human, cat, and mouse FLVCR, but are not conserved in closely related paralogs (Gene 286:203, 2002), which lack heme export function. We hypothesize they act as a temporary docking site for heme after it exits through the transport channel and prior to being picked up by HPXN in the circulation, and thus as a structural regulator of export efficiency. We anticipate that when heme binds to this site, the channel is held in the open-out position preventing additional heme from being transported. We are currently using site-directed mutagenesis to generate a mutant FLVCR lacking these two histidines and will test its export function. To our knowledge, comparable regulatory mechanisms have not been previously described for other MFS family members or for transport proteins more broadly. Since HPXN is present in a high concentration in human serum (17–25 μM, similar to transferrin, 22–31 μM), it may function physiologically to facilitate the recycling of heme-iron from macrophages after they ingest senescent red cells, in addition to its role as a scavenger protein for protection from internal bleeding or extravascular hemolysis. By extension, modulation of the serum HPXN concentration might ameliorate the anemia of chronic inflammation.

Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development

AbstractErythropoiesis, the essential process of hematopoietic stem cell development into erythrocytes, is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver, neonatal spleen, and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen, short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype, demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation, including hemoglobinization, cell condensation, and enucleation, and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells.

Multipotential differentiation ability of GATA-1-null erythroid-committed cells

GATA-1, a zinc finger transcription factor, has been believed to be indispensable for the survival of proerythroblasts. However, we found that GATA-1-null proerythroblasts could survive and proliferate on OP9 stroma cells in the presence of erythropoietin. Furthermore, myeloid and mast cells were induced from the GATA-1-null proerythroblasts by the stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), respectively, but lymphoid differentiation was not achieved by in vivo transfer. Thus, without activity of the transcription factor required for terminal differentiation, even relatively mature and committed cells proliferate continuously with the differentiation capacity to other lineages. Our data suggest that GATA-1 is a critical transcription factor to fix erythroid progenitors to the erythroid lineage.

Mechanisms of Erythroid Precursor Expansion in Response to Erythropoietin and Stem Cell Factor: Requirements for Cooperative Signal Transduction.

The adult bone marrow produces 2x1011 red blood cells daily to maintain the oxygen carrying capacity of peripheral blood. Certain pathologic conditions such as blood loss or hemolysis that result in increased oxygen demands lead to a physiologic bone marrow response characterized by increased rate of erythropoiesis and expansion of bone marrow proerythroblasts, the precursor cells that differentiate into mature red blood cells. We studied the mechanisms by which erythropoietin (EPO) and stem cell factor (SCF) regulate the expansion of primary human proerythroblasts. Using liquid cultures of peripheral blood CD34+ cells isolated from healthy volunteers, we generated uniform populations of transferrin receptor (CD71)+ human proerythroblasts. We established a serum-free culture model to study the effect of EPO and SCF on the survival and proliferative capacity of the cells. Primary proerythroblasts failed to survive in the presence of EPO or SCF alone, but exhibited marked synergistic proliferation in response to EPO plus SCF, exhibiting one log expansion in 5 days under serum-free conditions. Characterization of EPO receptor (EPOR) and SCF receptor (KIT)-mediated signal transduction in proerythroblasts revealed a requirement for EPOR, but not KIT, signaling for tyrosine phosphorylation of STAT5 (Tyr694), a downstream target for the cytoplasmic tyrosine kinase JAK2. MAP kinases ERK 1/2 were phosphorylated (Thr202/Tyr204) in response to either EPO or SCF alone, with phosphorylation of ERK1/2 induced predominantly by SCF. We found increased phosphorylation of ERK 1/2 when proerythroblasts were treated with both EPO and SCF. Phosphorylation of protein kinase B/Akt (Ser473), a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K), was observed following SCF treatment. Treatment with kinase inhibitors targeting JAK, PI3K and MAP kinase kinase (MEK1) during EPO and SCF stimulation revealed that JAK inhibitor AG490 attenuated STAT5 tyrosine phosphorylation, MEK inhibitor PD98059 abolished ERK 1/2 phosphorylation and the PI3K inhibitor LY294002 inhibited Akt phosphorylation. To determine the contribution of specific signaling pathways to synergistic proliferation of proerythroblasts in response to cooperative effects of EPO and SCF, we performed proliferation assays with increasing concentrations of each inhibitor (5 and 50 μM) or DMSO vehicle. We found significant, dose-dependent inhibition of proerythroblast proliferation in response to all three JAK, PI3K or MEK inhibitors (figure 1, *P<0.001 by ANOVA, n=6). We conclude that 1)- EPO or SCF alone fail to support proerythroblast survival, 2)- The cooperative and synergistic effects of EPO and SCF are required for the expansion of primary erythroid precursors, 3)- EPOR but not KIT signaling mediates tyrosine phosphorylation of STAT5 in proerythroblasts, 4)- Phosphorylation of Akt and ERK1/2 in proerythroblasts is mediated primarily by SCF, 5)- EPO and SCF together lead to increased phosphorylation of ERK 1/2, and 6)- The cooperative effect of EPO and SCF that mediates synergistic erythroid precursor expansion requires activation of multiple signaling pathways, including the JAK-STAT, PI3K and MAP kinase pathways. [Display omitted]