Epo Signaling Research Articles

In Vitro and in Vivo Abrogation of An Erythroid Iron Restriction Developmental Checkpoint by Isocitrate through a Novel Erythropoietin- Coupled Signaling Mechanism

Erythropoietin (Epo) signaling drives normal erythropoiesis by promoting the survival, proliferation and maturation of committed erythroid progenitor cells. Epo acts at an early stage in erythroid development, prior to the initiation of hemoglobin synthesis. Under conditions of iron restriction, erythroid progenitors become refractory to Epo, resulting in hypoplastic anemia. The resulting iron restriction checkpoint protects iron stores from depletion by preventing Epo-driven erythroid expansion and inappropriate iron utilization for hemoglobin synthesis. In addition to diminished body stores, defects in iron uptake or intracellular trafficking can also activate this checkpoint and contribute to Epo-refractory anemias, e.g. sideroblastic anemias. Our previous work using primary human hematopoietic cultures had implicated aconitase enzymes, which interconvert citrate and isocitrate, as critical regulators of the erythroid iron-restriction checkpoint. In those studies, supplying cells with isocitrate had completely abrogated the block in erythroid development caused by iron restriction. In the current studies, we examine the mechanism for isocitrate rescue of erythropoiesis in iron deprived human progenitors and determine the in vivo effects of isocitrate administration in mice with iron deficiency anemia. Initial experiments addressed whether the activity of isocitrate was due to its catabolism to yield ATP and succinyl CoA, a precursor of heme. Several independent findings argued against such a metabolic mechanism. Firstly, erythroid progenitors showed no changes in cellular [ATP] with iron deprivation −/+ isocitrate. Secondly, a bioactive analog of alpha-ketoglutarate, TaKG, failed to rescue erythropoiesis under iron deprivation. Thirdly, isocitrate promoted erythroid differentiation in progenitors with blockade in mitochondrial biogenesis, induced by chloramphenicol. In these last studies, isocitrate reversed chloramphenicol inhibition of glycophorin A and globin expression; exogenous hemin by contrast reversed only the inhibition of globin expression. The combination of isocitrate and hemin, however, showed strong synergy in the rescue of growth and globin expression in cholaramphenicol treated progenitors. Subsequent experiments tested the hypothesis that isocitrate functions as a second messenger in erythroid development. Accordingly, iron deprived erythroid progenitors exposed to a range of Epo levels (0.05–20 U/ml) underwent isocitrate treatment. Remarkably, isocitrate showed no rescue of iron deprived erythroid cultures with 0.05 U/ml Epo, a dose that still promotes erythroid differentiation in high iron cultures. Partial rescue was obtained with 0.2 U/ml Epo, and complete rescue with > 4.5 U/ml. Previous studies have suggested that Epo-mediated calcium signaling shows a strong dosage dependency, requiring relatively high doses of Epo to activate calcium influx. In human CD34+ progenitors exposed to 4.5 U/ml Epo for either 5 hours or 3 days, iron deprivation induced a drop in steady state intracellular calcium levels. Inclusion of isocitrate in the medium restored the intracellular calcium to the levels seen in the iron-replete cultures. Finally, the in vivo activity of isocitrate was assessed in a murine model of iron deficiency anemia. Strikingly, intraperitoneal injections of isocitrate (200 mg/kg/day for 5 days) significantly augmented the red cell counts in C57BL/6 weanlings subjected to a low iron diet: mean RBC of 9.3 ± 0.35 × 10e12 cells/liter for the isocitrate group compared to an average RBC of 7.4 ± 0.51 × 10e12 cells/liter for the saline control group (P=0.012, N = 6 for each group). Interestingly, this increase accompanied a parallel decrease in red cell mean corpuscular hemoglobin concentration. Taken together, our results suggest a role for isocitrate as a second messenger coupled to Epo signaling and potentially involved in intracellular calcium regulation. In vivo administration abrogates the iron restriction checkpoint on red cell production, as in ex vivo studies, but cannot drive hemoglobin synthesis in the face of limited iron stores. The ability of isocitrate to collaborate with hemin in overriding erythroid mitochondrial defects offers novel therapeutic avenues for sideroblastic anemias.

Role of mTOR Signaling in Erythropoiesis

Erythropoietin (Epo) signaling is required for differentiation of erythroid progenitors to mature red blood cells. Binding of Epo to its receptor activates Jak2, which in turn activates many signaling proteins including AKT, MAPK proteins and STATs. We have shown previously that AKT is required for Epo regulation of erythroid cell maturation; activated AKT complements Epo receptor signaling in JAK2-deficient fetal liver cells and supports erythroid cell differentiation. AKT functions by phosphorylating several proteins including FoxO3 and mTOR. AKT phosphorylation of FoxO3 represses FoxO3's activity, whereas AKT-dependant phosphorylation activates mTOR and its downstream target p70 S6 kinase (S6K). We have shown recently that FoxO3 is essential for the regulation of erythroid cell cycling, maturation, lifespan and anti-oxidant response (Marinkovic et al., JCI, 2007). Here we aimed at identifying other proteins in AKT signaling network that may regulate the maturation of erythroid progenitors. To address this, we inhibited several signaling pathways and analyzed their role in Epo-dependant maturation of freshly-isolated E14 fetal liver progenitors. As anticipated, blocking PI3-Kinase resulted in 60 % reduction of BFU-E- and CFU-E-derived colony formation and blocked the maturation of erythroid progenitors. Interestingly, blocking either p38 or ERK MAPK signaling showed 40% reduction in erythroid BFU-E- and CFU-E-derived colony formation. Surprisingly, blocking of mTOR signaling inhibited the formation of BFU-E- and CFU-E-derived colonies by 75 %. Further analysis by flow cytometry monitoring of cell surface markers CD71 and TER 119 showed that erythroid progenitor cell maturation could not proceed past early erythroblast stage when cells were cultured in the presence of rapamycin overnight. We confirmed that this block in differentiation was not due to apoptosis of erythroid cells. Since both FoxO3 and mTOR work downstream of AKT, we asked whether inhibition of mTOR has any impact on FoxO3 activity. Epo stimulation of freshly isolated bone marrow lineage-negative cells previously starved from cytokines showed a 2.3 fold increase in FoxO3 phosphorylation in the presence of rapamycin, suggesting cross talk between mTOR and FoxO3. Next, we investigated the effect of loss of FoxO3 on AKT/mTOR signaling in erythroid precursors. To address this, we prepared a population of bone marrow depleted from lineage-restricted cells and cultured under optimum erythroid conditions that generated 60% erythroblasts after 18 hours. Epo stimulation of FoxO3 null erythroid precursors led to hyperphosphorylation of Jak2, AKT, mTOR and S6K as compared to control cells. Since FoxO3 is critical for repression of reactive oxygen species (ROS), we evaluated the potential role of ROS in activating these proteins in FoxO3 mutant erythroid cells. In vitro treatment with ROS scavenger N-Acetyl-Cysteine (NAC) reduced significantly the hyper-phosphorylation of AKT, mTOR and S6K in FoxO3 null erythroid precursors in response to Epo. In addition, our results suggest that phosphorylation of JAK2 and its downstream signaling proteins AKT/mTOR/S6K in primary wild type erythroid precursor cells in response to Epo is mediated by ROS. Interestingly, ROS modulation of phosphorylation of mTOR/S6K was significantly stronger than that of AKT in response to Epo-stimulation of primary erythroid cells. Activation of AKT/mTOR/S6K is likely to mediate increased production of erythroid precursors observed in FoxO3 mutant mice (Marinkovic et al., JCI, 2007). Collectively these results indicate an important function for the AKT/mTOR/S6K signaling pathway in Epo-dependant erythropoiesis and suggest that cytokine-mediated production of ROS plays a critical role in the regulation of primary erythroid cell formation.

Role of the Erythropoietin Receptor in ETV6/RUNX1-Positive Acute Lymphoblastic Leukemia

We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias. ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols. We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup. Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.

Erythropoiesis-Stimulating Agent Use in Cancer: Preclinical and Clinical Perspectives

Erythropoiesis-stimulating agents (ESA) used for the treatment of chemotherapy-induced anemia in cancer patients have been associated with adverse outcomes of enhanced tumor progression and impaired survival in a series of recent clinical trials. As clinical practice guidelines for ESA administration in cancer patients have evolved to improve safety, the mechanisms underlying the adverse outcomes and whether ESAs exert direct and/or indirect effects in primary tumors to modulate tumor cell growth, survival, and chemoradiotherapy responses remain uncertain. Erythropoietin receptor (EpoR) expression in tumor cells has raised the simplistic possibility that Epo signaling mediated via a functional cellular receptor may contribute to tumor progression in a direct manner. However, Epo biology in cancer is likely to be complex and an interplay of multiple factors is potentially involved in the overall tumor response to exogenous Epo. Optimization of ESA use as an important supportive therapy modality in cancer patients, and further investigation of the role of Epo-EpoR in cancer biology will require a combination of carefully designed preclinical and clinical studies designed to ascertain not only the effect of ESA therapy on clinical outcomes such as tumor response, progression-free, and overall survival but also to investigate the potential effects of Epo on biomarkers of EpoR activation and factors related to tumor biology and chemoradiation responsiveness.

Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y 913) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y 913 rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y 1007/1008 in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y 913 (Y 913F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y 913 (Y 913E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y 913F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y 913E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y 939, a corresponding tyrosine residue with Y 913, negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.

Synergistic upregulation of erythropoietin receptor (EPO-R) expression by sense and antisense EPO-R transcripts in the canine lung

We previously found increased erythropoietin receptor (EPO-R) protein levels in vigorously growing canine lungs after pneumonectomy (PNX), suggesting a role for paracrine EPO signaling in lung growth and remodeling. Now we find that sense and antisense EPO-R transcripts (sEPO-R and asEPO-R, respectively) are concordantly up-regulated in the post-PNX remaining lung, leading to the hypothesis that sEPO-R and asEPO-R interactions enhance EPO signaling during lung growth. We cloned a canine asEPO-R cDNA, which is fully complementary to the sense strand of the EPO-R gene from 2.5kb 3' to the sense stop codon, and extends into the 5' UTR of the sEPO-R transcript. Both asEPO-R and sEPO-R transcripts colocalize with EPO-R protein in the same lung cells. In cultured human embryonic kidney (HEK293) cells, transfection with sEPO-R (+FLAG tag) cDNA alone increased EPO-R protein expression (anti-EPO-R and anti-FLAG). At constant sEPO-R cDNA levels, cotransfection with escalating asEPO-R cDNA further increased recombinant EPO-R protein expression. The asEPO-R transcript harbors two putative opening reading frames (ORFs). Separate transfection of each asEPO-R ORF cDNA resulted in differential stimulatory effects on EPO-R protein expression. We conclude that both sEPO-R and asEPO-R transcripts contribute to in vivo up-regulation of EPO-R protein expression in the post-PNX remaining lung. This demonstrates synergism between sense-antisense EPO-R transcripts in response to physiological stimulation in a robust model of induced lung growth.

Synergistic regulation of erythropoietin receptor (EPO‐R) expression by sense and antisense EPO‐R transcripts in the canine lung

We had found that EPO‐R protein is upregulated in growing canine lungs following pneumonectomy (PNX) (Am J Physiol 287:L1107, 2004). We also found both sense and anti‐sense EPO‐R transcripts (sEPO‐R and asEPO‐R) to be upregulated in post‐PNX remaining lungs. We hypothesized that sEPO‐R and asEPO‐R interactions modulate EPO signaling in lung growth. We cloned a canine asEPO‐R cDNA, fully complementary to the sense strand of EPO‐R gene starting from 2.5 kb 3′‐ to the sense stop codon, and extending into the 5′UTR of sEPO‐R transcript. The asEPO‐R transcript harbors two putative open reading frames but it is not clear if they translated. The asEPO‐R and sEPO‐R transcripts co‐localize to bronchiolar epithelium and alveolar capillary endothelium. In HEK 293 cells transfection with sEPO‐R (+FLAG tag) cDNA alone increased EPO‐R protein expression, detected by anti‐EPO‐R and anti‐FLAG. At a constant sEPO‐R cDNA level, co‐transfection with escalating levels of asEPO‐R cDNA further increased recombinant EPO‐R protein expression. Thus, both sEPO‐R and asEPO‐R transcripts contribute to in vivo EPO‐R upregulation in the remaining lung post‐PNX. These data demonstrate novel synergism between complementary sense‐antisense transcripts in response to physiological stimulation in a robust model of induced lung growth. Supported by NHLBI RO1 HL40070, 62873, 45716, 54060, DK48482, DK‐20543 and Simmons Family Foundation.

A proteomic analysis of CBA‐Pk‐1slc/Pk‐1slc mice with Red Blood Cell Type Pyruvate Kinase deficiency using 2DE together with MALDI–TOF‐MS analysis

SLC3 is a Friend erythroleukemic cells line from the Pk‐1slc mouse erythrocyte pyruvate kinase(PK)deficiency. SLC3 undergo spontaneous apoptosis leads to ineffective erythropoiesis. It was showed that glycolytic inhibition by PKLR gene mutation augmented oxidative stress, leading to the activation of HIF‐1 as well as downstream proapoptotic gene expression. We studied proteomic response of SLC3 was quantitatively analyzed two‐dimensional gel electrophoresis (2DE) followed by identification with matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF‐MS). We detected significant differences between SLC3 and a control Friend cell. 72% of the total proteins studied were significantly down regulated in SLC3 and only 28% of proteins were up regulated. Several membrane proteins are significantly down‐regulated in SLC3 may cause reduced stability and integrity of PK deficient RBC. Oxidative stress proteins in SLC3 mice were up‐regulated and antioxidant proteins were down regulated. These proteins related to generation of ROS. For the refolding of mutant protein, molecular chaperon is expectedly unregulated in SLC3. Upregulation of two chaperons are presumably an adapted reaction in SLC3. Heat‐shock protein (HSP70) and mortalin, A novel mediator of erythropoietin signaling. We speculated that down‐regulation of mortalin might be attributable to disturbance of EPO signaling, resulting in premature death of cells. The proteomic approach makes an important contribution to characterizing the proteome of the SLC3.

Dysregulated Signaling Networks in Chronic Myeloproliferative Disorders: Co-Operation of JAK2 and Src Family Kinases (SFK)

The JAK2-STAT5 pathway plays an key role in chronic MPD (cMPD) with the JAK2 V617F mutation. However, JAK2 also initiates several second messenger pathways, including the Ras-ERK and PI3K-AKT pathways. Since these essential second messenger pathways are also regulated by SFK activity, and crosstalk between JAK and SFK has been demonstrated, we investigated the potential SFK-dependent regulation of STAT5, Ras-ERK and PI3K-AKT in primary cells from patients with cMPD and myeloid cell lines with the JAK2 V617F mutation (HEL) or Epo-dependent (UT-7/Epo). In primary cells from JAK2 V617F patients, we show that SFK activity is required for optimal JAK2 induced phospho (p)-STAT5, nuclear targeting of p-STAT5, and STAT5-DNA binding. Both endogenous and Epo-induced colony formation in methylcellulose by peripheral blood cells derived from polycythemia vera (PV) patients was markedly suppressed with the SFK inihibitor dasatinib; endogenous colony formation was abrogated while both the number and size of Epo-induced colonies was markedly reduced (> 70% inhibition at 1 uM), suggesting that despite of the presence of activating JAK2 V617F in primary cells, SFK activity is required for optimal proliferation. Interestingly, a similar observation was also made in a PV patient lacking the JAK2 V617F mutation. When HEL or Epo-stimulated UT-7/Epo cells were treated with a JAK inhibitor (P6), there was significant inhibition of p-STAT5 induction and second messenger pathways, demonstrating the importance of this proximal kinase. Inhibition of JAK2 did not affect SFK activation however, suggesting that SFK may lie upstream of JAK2. Interestingly, when SFK activity was suppressed (PP2 or dasatinib), a marked inhibition of p-STAT5 (and STAT5 DNA-binding), p-AKT, p-ERK, p-90RSK, p-S6 ribosomal protein(rp), and global protein tyrosine phosphorylation was observed in cell lines and primary cells. These findings suggest that SFK activity is involved in the most proximal aspects of Epo signaling and facilitates JAK2 activation of key second messenger pathways essential for proliferation, differentiation, and survival. Since JAK2 and SFK coordinately regulate multiple second messenger pathways, we next examined the relative importance of each by specifically targeting either the Ras-ERK pathway (MEK inhibition) or the PI3K-AKT pathway (PI3K and mTOR inhibition) downstream from JAK2 and SFK in both primary cells and cell lines. Using intracellular phospho-flow cytometry and immunoblotting, we show that the SFK-dependent Ras-ERK-RSK axis can directly induce p-S6rp, a key component in translational control and cell cycle progression. To determine whether this finding can be reproduced in vivo, we examined p-S6rp expression in bone marrow biopsies from PV patients (n=16); a marked hyperphosphorylation S6rp was consistently seen in patients' megakaryocytes as compared to control specimens obtained from reactive processes, lymphoma staging, CMML, or even a case of hypoxia-induced polycythemia (all showing low/absent p-S6rp). Simultaneous staining for p-mTOR revealed that the level of p-mTOR was not increased. In sum, our findings suggest that SFK activation is necessary for optimal signal transduction along of the JAK2-STAT5 axis. In addition, the Ras-ERK-RSK pathway, requiring both JAK and SFK, may play a key role in S6rp hyperphosphorylation leading to increased cell cycling. Both findings point towards potential therapeutic targets, along with JAK2, for treating cMPD.

Biological Significance of Erythropoietin and Erythropoietin Receptor Expression by Human Ovarian Cancer Cells.

Erythropoietin (Epo) is the primary regulator of erythroid cell proliferation and differentiation mediated through its specific binding to the Epo receptor (EpoR). Epo also is known to have non-hematopoietic actions, including the promotion of wound healing, tissue protection and others. Accumulating evidence demonstrating both Epo and EpoR expression in many types of human cancers has raised concern about the clinical use of Epo and other erythropoiesis stimulating agents (ESAs) in cancer-related anemia, since the presence of functional EpoR on the tumor cells suggests the potential for an adverse effect of Epo treatment by enhancing cancer growth and progression. Previously, we demonstrated that A2780 ovarian cancer cells express the EpoR gene and that long-term Epo treatment of these cells renders them resistant to paclitaxel. We now report the characterization and biological significance of Epo and EpoR expression by four ovarian cancer cell lines. Using semi-quantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we demonstrated that A2780, CaOV, SKOV, and OVCAR-3 ovarian cancer cell lines examined express Epo and EpoR at the mRNA level. We demonstrated EpoR protein expression both by western blotting and by immunofluorescence, and demonstrated biologically active Epo protein expression by quantitative in vitro bioassay of the cell culture supernatants. The EpoR on A2780 cells are functional, since Epo stimulation of the cells resulted in a time-dependent 5-fold increase in phosphorylation of Erk1/2 that reached a peak in 10 min and then returned to the basal level. None of the cell lines exhibited a short-term (3-day) growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to the cell culture resulted in partial growth inhibition of A2780 cells that was reversed by addition of excess Epo, consistent with an autocrine/paracrine mechanism of Epo growth enhancement of these cells. We also found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting enhanced Epo signaling, evidenced by a 20-fold increase in phosphorylation levels of Erk1/2. This phenotype was sustained even after the removal of Epo, suggesting a mechanism underlying the paclitaxel resistance developed after long-term Epo treatment. Our findings have implications for the clinical use of recombinant human Epo and other ESAs to correct and/or prevent anemia in ovarian and, potentially, other cancer patients. They also suggest that endogenous Epo and the EpoR may serve as novel therapeutic targets in the treatment of cancer patients.

Erythropoietin treatment of human ovarian cancer cells results in enhanced signaling and a paclitaxel‐resistant phenotype

Erythropoietin (Epo), a glycoprotein hormone that is the principal regulator of erythropoiesis, is known to act also on nonhematopoietic cell types. Epo receptors have been reported on several normal and neoplastic human cells and tissues, including ovarian cancer cells. We found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting both enhanced Epo signaling, evidenced by increased peak levels of phospho-Erk1/2 and increased paclitaxel resistance. This phenotypic effect was specific for paclitaxel, since no change in cisplatin or carboplatin sensitivity was observed. In addition, the change in phenotype was stable, even after the removal of Epo. Measurement of mono- and oligonucleosome formation revealed that long-term Epo treated A2780 cells exhibited markedly less apoptosis than nonerythropoietin treated cells at essentially all concentrations of paclitaxel tested. Western blot analyses revealed that the long-term Epo treated cells had significantly reduced expression of apoptosis-related proteins Bcl-2 and Bcl-10. These findings may have implications for the clinical use of recombinant human Epo and other erythropoiesis stimulating agents to correct anemia in paclitaxel-treated cancer patients.

Hypoxia alters progression of the erythroid program

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Functional conservation of erythropoietin signaling in zebrafish

Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.

Endogenous Erythropoietin Signaling Is Required for Normal Neural Progenitor Cell Proliferation

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

β-Trcp mediates ubiquitination and degradation of the erythropoietin receptor and controls cell proliferation

AbstractControl of intensity and duration of erythropoietin (Epo) signaling is necessary to tightly regulate red blood cell production. We have recently shown that the ubiquitin/proteasome system plays a major role in the control of Epo-R signaling. Indeed, after Epo stimulation, Epo-R is ubiquitinated and its intracellular part is degraded by the proteasome, preventing further signal transduction. The remaining part of the receptor and associated Epo are internalized and degraded by the lysosomes. We show that β-Trcp is responsible for Epo-R ubiquitination and degradation. After Epo stimulation, β-Trcp binds to the Epo-R. This binding, like Epo-R ubiquitination, requires Jak2 activation. The Epo-R contains a typical DSG binding sequence for β-Trcp that is highly conserved among species. Interestingly, this sequence is located in a region of the Epo-R that is deleted in patients with familial polycythemia. Mutation of the serine residue of this motif to alanine (Epo-RS462A) abolished β-Trcp binding, Epo-R ubiquitination, and degradation. Epo-RS462A activation was prolonged and BaF3 cells expressing this receptor are hypersensitive to Epo, suggesting that part of the hypersensitivity to Epo in familial polycythemia could be the result of the lack of β-Trcp recruitment to the Epo-R.

Unrestrained erythroblast development in Nix −/− mice reveals a mechanism for apoptotic modulation of erythropoiesis

Normal production of RBCs requires that the antiapoptotic protein Bcl-xl be induced at end stages of differentiation in response to erythropoietin (Epo) signaling. The critical proapoptotic pathways inhibited by Bcl-xl in erythroblasts are unknown. We used gene targeting in the mouse to evaluate the BH3-only factor Nix, which is transcriptionally up-regulated during Epo-stimulated in vitro erythrocyte differentiation. Nix null mice are viable and fertile. Peripheral blood counts revealed a profound reticulocytosis and thrombocytosis despite normal serum Epo levels and blood oxygen tension. Nix null mice exhibited massive splenomegaly, with splenic and bone marrow erythroblastosis and reduced apoptosis in vivo during erythrocyte maturation. Hematopoietic progenitor populations were unaffected. Cultured Nix null erythroid cells were hypersensitive to Epo and resistant to apoptosis stimulated by cytokine deprivation and calcium ionophore. Transcriptional profiling of Nix null spleens revealed increased expression of cell cycle and erythroid genes, including Bcl-xl, and diminished expression of cell death and B cell-related genes. Thus, cell-autonomous Nix-mediated apoptosis in opposition to the Epo-induced erythroblast survival pathway appears indispensable for regulation of erythrocyte production and maintenance of hematological homeostasis. These results suggest that physiological codependence and coordinated regulation of pro- and antiapoptotic Bcl2 family members may represent a general regulatory paradigm in hematopoiesis.

Spi-1/PU.1 participates in erythroleukemogenesis by inhibiting apoptosis in cooperation with Epo signaling and by blocking erythroid differentiation

AbstractOverexpression of the transcription factor Spi-1/PU.1 in mice leads to acute erythroleukemia characterized by a differentiation block at the proerythroblastic stage. In this study, we made use of a new cellular system allowing us to reach graded expression of Spi-1 in preleukemic cells to dissect mechanisms of Spi-1/PU-1 in erythroleukemogenesis. This system is based on conditional production of 1 or 2 spi-1–interfering RNAs stably inserted into spi-1 transgenic proerythroblasts. We show that Spi-1 knock-down was sufficient to reinstate the erythroid differentiation program. This differentiation process was associated with an exit from the cell cycle. Evidence is provided that in the presence of erythropoietin (Epo), Spi-1 displays an antiapoptotic role that is independent of its function in blocking erythroid differentiation. Apoptosis inhibited by Spi-1 did not involve activation of the Fas/FasL signaling pathway nor a failure to activate Epo receptor (EpoR). Furthermore, we found that reducing the Spi-1 level yields to ERK dephosphorylation and increased phosphorylation of AKT and STAT5, suggesting that Spi-1 may affect major signaling pathways downstream of the EpoR in erythroid cells. These findings reveal 2 distinct roles for Spi-1 during erythroleukemogenesis: Spi-1 blocks the erythroid differentiation program and acts to impair apoptotic death in cooperation with an Epo signaling.

Expression of Erythropoietin and Erythropoietin Receptor in Vestibular Schwannoma

Background: Expression of erythropoietin (Epo) is controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α). Epo signaling via erythropoietin receptor (EpoR) results in stimulation of cell proliferation and upregulation of the antiapoptotic protein bcl-2 followed by inhibition of apoptosis. Epo has previously been demonstrated to be associated with Schwann cell proliferation and a recent clinical case report suggested a role in vestibular schwannoma growth.

Expression of Erythropoietin and Erythropoietin Receptor in Vestibular Schwannoma

Background: Expression of erythropoietin (Epo) is controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α). Epo signaling via erythropoietin receptor (EpoR) results in stimulation of cell proliferation and upregulation of the antiapoptotic protein bcl-2 followed by inhibition of apoptosis. Epo has previously been demonstrated to be associated with Schwann cell proliferation and a recent clinical case report suggested a role in vestibular schwannoma growth.

Molecular Pathogenesis and Therapy of Polycythemia Induced in Mice by JAK2 V617F

BackgroundA somatic activating mutation (V617F) in the JAK2 tyrosine kinase was recently discovered in the majority of patients with polycythemia vera (PV), and some with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis. However, the role of mutant JAK2 in disease pathogenesis is unclear.Methods and FindingsWe expressed murine JAK2 WT or V617F via retroviral bone marrow transduction/transplantation in the hematopoietic system of two different inbred mouse strains, Balb/c and C57Bl/6 (B6). In both strains, JAK2 V617F, but not JAK2 WT, induced non-fatal polycythemia characterized by increased hematocrit and hemoglobin, reticulocytosis, splenomegaly, low plasma erythropoietin (Epo), and Epo-independent erythroid colonies. JAK2 V617F also induced leukocytosis and neutrophilia that was much more prominent in Balb/c mice than in B6. Platelet counts were not affected in either strain despite expression of JAK2 V617F in megakaryocytes and markedly prolonged tail bleeding times. The polycythemia tended to resolve after several months, coincident with increased spleen and marrow fibrosis, but was resurrected by transplantation to secondary recipients. Using donor mice with mutations in Lyn, Hck, and Fgr, we demonstrated that the polycythemia was independent of Src kinases. Polycythemia and reticulocytosis responded to treatment with imatinib or a JAK2 inhibitor, but were unresponsive to the Src inhibitor dasatinib.ConclusionsThese findings demonstrate that JAK2 V617F induces Epo-independent expansion of the erythroid lineage in vivo. The fact that the central erythroid features of PV are recapitulated by expression of JAK2 V617F argues that it is the primary and direct cause of human PV. The lack of thrombocytosis suggests that additional events may be required for JAK2 V617F to cause ET, but qualitative platelet abnormalities induced by JAK2 V617F may contribute to the hemostatic complications of PV. Despite the role of Src kinases in Epo signaling, our studies predict that Src inhibitors will be ineffective for therapy of PV. However, we provide proof-of-principle that a JAK2 inhibitor should have therapeutic effects on the polycythemia, and perhaps myelofibrosis and hemostatic abnormalities, suffered by MPD patients carrying the JAK2 V617F mutation.