EpoR Expression Research Articles

PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells

Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and γ-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.

Abstract 238: Role of Erythropoietin Signaling in the Biology of Mouse Cardiac Progenitor Cells

Erythropoietin (Epo) was shown to improve cardiac function following ischemia reperfusion mainly via neo-angiogenesis and anti-apoptotic mechanisms. We found EpoR expression to be particularly high in adult cardiac progenitor cells (CPCs). Thus, we reasoned that Epo may play a role in the biology of these cells. We isolated CPCs from adult C57BL/6 hearts by enzymatic digestion and filtration (pore size: 30 µm). By means of immunofluorescence microscopy (IF) and flow cytometry (FC) we analyzed EpoR expression in the CPCs. 24±3% of the investigated cardiac cells were positive for EpoR with 3±2% of these being c-kit+ and 28%±2% Sca-1+. 52% of the EpoR+ cells expressed endothelial cell markers (40±2% CD34+, 9±2% FLK1+). 42±4% expressed myocyte markers (αMHC+, cTNT+). IF revealed a progenitor-like population with immature cell morphology and proliferation potential (ki67+). Cell cycle analysis showed an enrichment of αMHC+ EpoR+ cells in S and G2 phase (49±7%, n=3) as compared to the αMHC- EpoR- population (13±3%, n=3). Moreover, we tested the effect of Epo in the biology of these CPCs in vitro. At d14 we observed a two-fold increase of GATA4+ and cTnT+ cardiac cells in the co-cultures treated with Epo (n=3). CPC cycle arrest abrogated the aforementioned effects, suggesting that Epo influences mainly CPC proliferation. Finally, we tested the potential of Epo to protect against ischemia by inducing the proliferation of these αMHC+ CPCs in vivo in a myocardial infarction (MI) model. 4 weeks post MI, echocardiography did not reveal a significant functional improvement of the Epo receiving mice (2x, 2U/g Epo i.p). Nevertheless, FC analysis of the progenitor pool showed a significant augmentation of αMHC+ and cTnT+ cells (Sham: 19±3% vs Epo 35±3%, n=5; MI: 10.6±2.3%, n=6 vs Epo 20.3±1.9%, n=8). These data suggest an activation of myogenic progenitors by Epo, despite the lack of apparent regeneration under the investigated conditions. In conclusion, we found that EpoR is expressed in a putative cardiomyogenic progenitor cell pool in the adult heart. Epo drives their proliferation in vitro and in vivo even upon acute cardiac injury. We are currently investigating the long-term consequences of the observed progenitor cell activation in models of chronic ischemic injury.

EPO reverses defective wound repair in hypercholesterolaemic mice by increasing functional angiogenesis

This study aims to elucidate the effect of erythropoietin (EPO) on the microcirculation during wound healing in mice genetically depleted of apolipoprotein E (ApoE(-/-)). The skinfold chamber in mice was used for intravital microscopy, whereby an incisional wound was created within the chamber. Animals received Recormon(®) 1000 U kg(-1) body weight (BW) intra-peritoneally (i.p.) at day 1, 3, 5, 7, 9 and 11 post-wounding at a concentration of 100 Uml(-1) (n=42). Normal healing and vehicle-treated wild type animals (WT) served as controls. The microcirculation of the wound was analysed quantitatively in vivo using epi-illumination intravital fluorescence microscopy. Microtomography (micro-CT) analysis of casted wound microvessels was performed allowing three-dimensional (3D) histomorphometric analysis. Tissue samples were examined ex vivo for wound scoring and for expression analysis of EPO-Receptor (Epo-R) and endothelial nitric oxide synthase (eNOS). Upon EPO treatment, the total wound score in ApoE(-/-) mice was increased by 23% on day 3, by 26% on day 7 and by 18% on day 13 when compared to untreated ApoE(-/-) mice (all P<0.05 vs. vehicle). Improved wound healing was accompanied with a significant increase of functional angiogenetic density and angiogenetic red blood cell perfusion on days 5, 7, 9 and 11 post-wounding. 3D histomorphometric analysis revealed an increase of vessel thickness (1.7-fold), vessel volume (2.4-fold) and vessel surface (1.7-fold) (all P<0.05 vs. vehicle). In addition, improved wound healing was associated with enhanced Epo-R expression (4.6-fold on day 3 and 13.5-fold on day 7) and eNOS expression (2.4-fold on day 7) (all P<0.05 vs. vehicle). Our data demonstrate that repetitive systemic EPO treatment reverses microvascular dysfunction during wound healing in hypercholesterolaemic mice by inducing new vessel formation and by providing the wound with more oxygen.

Abstract 278: Signaling Pathways Involved in the Cytoprotective Effects of Erythropoietin and Erythropoietin Derivatives in Ischemic Human Myotubes

Objectives: Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously shown presence of the EpoR in human skeletal muscle. Here we aim to show the direct interaction of the EpoR with the βcR. Further, we investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish the role of PI3K/Akt pathway in protecting cells from apoptosis. Methods: Ethical approval and informed consent was obtained for gastrocnemius biopsies. Immunohistochemistry and western blot analysis were performed to demonstrate expression of EpoR and βcR in human skeletal muscle. Co-immunoprecipitation (Co-IP) was performed to demonstrate heterodimerisation of the EpoR with βcR. Human myoblasts were isolated from muscle biopsies to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitor, wortmannin, was then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results: The presence of both EpoR and βcR in human skeletal muscle has previously been demonstrated. We have now confirmed heterodimerisation of EpoR-βcR complex by Co-IP. Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of wortmannin, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Further, a reduction in apoptosis was associated with an increase in phosphorylated-Akt on western blot analysis. Conclusion: We have demonstrated EpoR-βcR heteroreceptor in human skeletal muscle. The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

Location and the functionality of erythropoietin receptor(s) in A2780 cells

Erythropoietin (Epo) is a critical regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of a recombinant protein, it is widely used to treat various forms of anemia, including that associated with cancer and with the myelosuppressive effects of chemotherapy. Studies of ovarian cancer cell lines have demonstrated the presence of the Epo receptor (EpoR), but there are disagreements regarding its localization and functionality in these cells. Using fluorescence microscopy, we were not able to identify the EpoR on the surface of A2780 cells, in contrast to the positive control K562 cells. Flow cytometry did reveal a weak surface EpoR signal in A2780 cells. Interestingly, most of the EpoR in A2780 cells was found in the cytoplasm, more abundantly as an intracellular membrane-associated protein than a soluble one. Silencing EpoR expression by lentiviral-mediated shRNA resulted in reduced A2780 proliferation as well as reduction in Epo-induced phosphorylation of Erk1/2. Our findings provide important insights into the biology of the EpoR in ovarian cancer cells.

Impact of increased erythropoietin receptor expression and elevated serum erythropoietin levels on clinicopathological features and prognosis in renal cell carcinoma

Erythropoietin (EPO) expression and EPO receptor (EpoR) expression have been demonstrated in various malignant tumors. EPO-EpoR signaling can activate several downstream signal transduction pathways that enhance tumor aggressiveness. The present study was undertaken to evaluate the impact of overexpression of EpoR and elevated serum EPO (sEPO) levels on the clinicopathological features and prognosis of patients with renal cell carcinoma (RCC). EpoR expression was evaluated immunohistochemically in 56 patients. Tumors with a staining intensity greater than that of surrounding proximal tubules were defined as tumors with high EpoR expression. The association between EpoR expression levels and various clinicopathological factors was analyzed. sEPO levels were determined in 138 patients and its correlation to clinicopathological factors was also analyzed, and EpoR expression was determined in surgical specimens removed from 47 of those 138 patients. Patients with high EpoR expression and patients with sEPO elevation had clinicopathological features less favorable than those of other patients. Tumors demonstrating high EpoR expression had a significantly higher number of Ki-67-positive cells compared to those with low EpoR expression. Tumor assemblies in microvessels demonstrated high EpoR expression. Patients whose tumors demonstrated high EpoR expression and those with sEPO elevation had a significantly lower survival rate compared to other patients, and patients with both high EpoR expression and sEPO elevation had an extremely poor prognosis. Microvascular invasion was an independent factor associated with sEPO elevation, suggesting that EPO-EpoR signaling might be important in RCC metastasis. EPO-EpoR signaling may be involved in tumor growth and progression in RCC and the combination of EpoR expression and sEPO levels may effectively predict clinical outcome.

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase

The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-κB activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP+EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-l-arginine methyl ester (l-NAME) simultaneously with EPO administration (CLP+EPO+l-NAME). A fifth group (CLP+EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP+EPO rats presented significantly higher inulin clearance than did CLP and CLP+EPO+l-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP+EPO rats; and inulin clearance was significantly higher in CLP+EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP+EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-α activation, NF-κB activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-κB downregulation.

Targeted Near-Infrared Imaging of the Erythropoietin Receptor in Human Lung Cancer Xenografts

The putative presence of the erythropoietin receptor (EpoR) on human cancer cells has given rise to controversial discussion about the use of recombinant human erythropoietin (rhuEpo) for treatment of patients with chemotherapy-induced anemia. In vivo analysis of the EpoR status in tumors could help in elucidating the role of erythropoietin in cancer. Thus, the aim of this study was to develop a targeted EpoR probe for the investigation of EpoR expression in human lung cancer xenografts by fluorescence-mediated tomography. Epo-Cy5.5 was generated by coupling Cy5.5 to rhuEpo. In vitro binding assays were performed using the EpoR-positive non-small cell lung cancer (NSCLC) cell lines A549 (lower EpoR expression) and H838 (higher EpoR expression), the EpoR-negative cell line H2030, and EpoR/EGFP-overexpressing HeLa cells. In vivo specificity of Epo-Cy5.5 was confirmed by competition analyses using micro-CT/fluorescence-mediated tomography fusion imaging. Biodistribution was analyzed over 50 h after injection. Binding of Epo-Cy5.5 was validated on tumor cryosections. After intravenous injection, the probe was rapidly cleared from the circulation. An accumulation was observed in liver and kidneys, with a maximum at 7 h after injection followed by a decline, indicating renal excretion. Almost constant accumulation of Epo-Cy5.5 was found in bone marrow and tumors, indicating specific receptor binding. The probe allowed the discrimination between H838 with higher EpoR expression (89.54 ± 15.91 nM at 25 h) and A549 tumors with lower EpoR expression (60.45 ± 14.59 nM at 25 h, P < 0.05). Tumor accumulation of Epo-Cy5.5 could be significantly reduced by adding unlabeled rhuEpo (P < 0.05 at 4, 7, and 24 h). In vitro validation confirmed specific binding of Epo-Cy5.5 to the tumor cells, and this binding correlated with the EpoR expression level. Binding was also observed on endothelial cells. Vessel density and Epo-Cy5.5 binding on endothelial cells were comparable. Epo-Cy5.5 allows the longitudinal analysis of EpoR expression in tumors and thereby can investigate the influence of erythropoietin on EpoR expression, tumor growth, and angiogenesis.

Effects of continuous erythropoietin receptor activator in sepsis-induced acute kidney injury and multi-organ dysfunction.

BackgroundDespite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI.MethodsRats were randomized into three groups: control; CLP; and CLP+CERA (5 µg/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting—to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-κB)—and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1β, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection.ResultsPretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-κB was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats.ConclusionCERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

The effect of erythropoietin on normal and neoplastic cells

Erythropoietin (Epo) is an essential hormone that binds and activates the Epo receptor (EpoR) resident on the surface of erythroid progenitor cells, thereby promoting erythropoiesis. Recombinant human erythropoietin has been used successfully for over 20 years to treat anemia in millions of patients. In addition to erythropoiesis, Epo has also been reported to have other effects, such as tissue protection and promotion of tumor cell growth or survival. This became of significant concern in 2003, when some clinical trials in cancer patients reported increased tumor progression and worse survival outcomes in patients treated with erythropoiesis-stimulating agents (ESAs). One of the potential mechanisms proffered to explain the observed safety issues was that functional EpoR was expressed in tumors and/or endothelial cells, and that ESAs directly stimulated tumor growth and/or antagonized tumor ablative therapies. Since then, numerous groups have performed further research evaluating this potential mechanism with conflicting data and conclusions. Here, we review the biology of endogenous Epo and EpoR expression and function in erythropoiesis, and evaluate the evidence pertaining to the expression of EpoR on normal nonhematopoietic and tumor cells.

Biomarkers Expression in Cervical Cancer and High Grade Squamous Intraepithelial Lesions

Objectives: The finding of new prognostic factors in human cervix cancer is necessary to improve present conventional treatments. The aim of the present study was to determine the expression and evaluate the prognostic value of hypoxia-inducible factor-1(HIF-1α), vascular endothelial growth factor (VEGF) and eritropoyetin receptor (EpoR) in cervix cancer stages IIA-IIB and in preinvasive high grade squamous intraepithelial lesions (HSIL) Methods: The study included 70 patients with cervix cancer, FIGO stages IIA-IIB, 28 patients with HSIL and normal cervix (n = 28). HIF-1α, VEGF and EpoR expression were analyzed in tissue samples by immunohistochemistry using commercial antibodies. Expression and overexpression of the tumor markers were quantified according to German Immunoreactive Score. Results: HIF-1α, EpoR and VEGF overexpression was detected in 30%, 37% and 51% of cancer patients respectively. Patients with HSIL showed enhanced expression only of EpoR and VEGF (39.2% and 71.4%) while VEGF was overexpressed in 21% of the specimen. No correlation was found between VEGF and EpoR with disease-free overall survival (OS), tumor recurrences or prognostic factors. Only overexpression of HIF-1 was associated with less median survival measured up to 24 months, unless it was not maintained a long time. Conclusion: Although any of the markers could be considered as independent prognostic factor for cervix cancer patients, our data showed a significant increase in their expression from the premalignant lesion up to the invasive stages of tumor progression.

Erythropoietin promotes oligodendrogenesis and myelin repair following lysolecithin-induced injury in spinal cord slice culture

Here, we sought to delineate the effect of EPO on the remyelination processes using an in vitro model of demyelination. We report that lysolecithin-induced demyelination elevated EPO receptor (EpoR) expression in oligodendrocyte progenitor cells (OPCs), facilitating the beneficial effect of EPO on the formation of oligodendrocytes (oligodendrogenesis). In the absence of EPO, the resultant remyelination was insufficient, possibly due to a limiting number of oligodendrocytes rather than their progenitors, which proliferate in response to lysolecithin-induced injury. By EPO treatment, lysolecithin-induced proliferation of OPCs was accelerated and the number of myelinating oligodendrocytes and myelin recovery was increased. EPO also enhanced the differentiation of neural progenitor cells expressing EpoR at high level toward the oligodendrocyte-lineage cells through activation of cyclin E and Janus kinase 2 pathways. Induction of myelin-forming oligodendrocytes by high dose of EPO implies that EPO might be the key factor influencing the final differentiation of OPCs. Taken together, our data suggest that EPO treatment could be an effective way to enhance remyelination by promoting oligodendrogenesis in association with elevated EpoR expression in spinal cord slice culture after lysolecithin-induced demyelination.

Erythropoietin produced by the retina: its role in physiology and diabetic retinopathy

Erythropoietin (Epo) is the principal regulator of erythropoiesis by inhibiting apoptosis and by stimulating the proliferation and differentiation of erythroid precursor cells. However, Epo also performs extra-erythropoietic actions of which the neuroprotective effects are among the most relevant. Apart from kidney and liver, Epo is also produced by the brain and the retina. In addition, Epo receptor (Epo-R) expression has also been found in the brain and in the retina, thus suggesting an autocrine/paracrine action which seems essential for the physiological homeostasis of both brain and retina. In this review, we will give an overview of the current concepts of the physiology of Epo and will focus on its role in the retina in both normal conditions and in the setting of diabetic retinopathy. Finally, the reasons as to why Epo could be contemplated as a potential new treatment for the early stages of diabetic retinopathy will be given.

Megakaryocytes Do Not Express Erythropoietin Receptor (EpoR) and Do Not Respond to Recombinant Human Erythropoietin (rHuEpo) Stimulation

Abstract 4783Recently, clinical studies were conducted in chronic kidney disease of type 2 diabetes patients that suggested an increased risk of stroke in patients receiving Erythropoiesis Stimulating Agents (ESAs) compared to placebo. One proposed explanation is that megakaryocytes and/or platelets express EpoR and respond to ESA challenge. We evaluated the underlying and untested assumption that megakaryocytes or platelets express a functional EpoR using flow cytometry assays developed to detect cell surface EpoR using validated EpoR-specific antibodies. Additionally, in order to investigate whether megakaryocytes/platelets are responsive to ESAs, the activation status of downstream signaling proteins was evaluated after stimulation with ESAs or vehicle control.Whole bone marrow aspirates were collected from healthy volunteers and bone marrow mononuclear cells (BMMCs) isolated by Ficoll separation (n=25). Megakaryocytes were isolated from BMMCs by using CD61 immuno-magnetic bead capture and analyzed by flow cytometry, gating for megakaryocytes using viability, CD41a expression and polyploidy. Platelets were analyzed in peripheral blood from healthy volunteers (n=10) using CD41a as marker.To measure EpoR expression an EpoR-specific monoclonal antibody was used for flow cytometry analysis in both platelets and megakaryocytes that were isolated and gated as described above. Megakaryocytes and platelets were stimulated with rHuEpo (1, 10, and 300 U/mL for 5 and 30 min) and induction of pSTAT5 was measured by intracellular flow cytometry using phospho-specific antibodies. In addition, platelets (CD41a+) were assessed for EpoR expression and functional response to recombinant human erythropoietin (rHuEpo) challenge. Stimulation with thrombopoietin (TPO) was used as a positive control for pSTAT5 induction in megakaryocytes and platelets.While robust EpoR expression was, as expected, observed consistently in UT7/Epo control cells and absent in CMK negative control cells (fold over control – UT7/Epo 12.78 ± 5.03; CMK 0.77 ± 0.22 mean ± 95%CI), no significant expression of EpoR was observed on CD41a+/polyploid megakaryocytes (fold over control − 1.006 ± 0.03; mean ± 95%CI). No activation of downstream signaling (pSTAT5) was detected in megakaryocytes in any of the samples stimulated with rHuEpo at any of the concentrations or timepoints analyzed, while robust pSTAT5 was detected in the UT7/Epo control cell lines. CMK cells were included as a negative control and demonstrated an absence of pSTAT5 induction in response to rHuEpo while a robust induction to TPO was observed (fold over control – UT7/Epo 12.11 ± 7.86; CMK 1.05 ± 0.03; mean ± 95%CI). Similarly, no significant EpoR expression was detected on CD41a+ platelets while the control, c-Mpl expression, was significant (fold over control – EpoR 1.11 ± 0.019; c-Mpl 2.936 ± 0.71; mean ± 95%CI) and no activation of downstream signaling (pSTAT5) was detected in platelets in any of the samples stimulated with rHuEpo at all concentrations and timepoints analyzed. The lack of EpoR expression on megakaryocytes was confirmed by laser scanning cytometric analysis in patient samples.A robust/sensitive platform was developed to profile biologically relevant EpoR expression and function in megakaryocytes and platelets. The assays for EpoR expression and function were sufficiently sensitive to allow characterization during in-vitro differentiation of erythroid progenitors. Using this platform we demonstrated a lack of EpoR expression and functional response to rHuEpo on megakaryocytes and platelets isolated from human bone marrow (CD41a+/CD61+/polyploid). Disclosures:Loberg:Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Manoukian:Amgen: Employment, Equity Ownership. Wang:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Sable:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership. McCaffery:Amgen: Employment, Equity Ownership.

Lenalidomide Upregulates Erythropoietin Receptor Expression in Hematopoietic Progenitors by Modulating Receptor Turnover

Abstract 2382The erythropoietin receptor (EpoR) is a type 1 cytokine receptor and key effector of Epo signaling. Maturation and shuttling of receptor to the plasma membrane is dependent on its association with Janus Kinase 2 (JAK2). Upon ligand binding, EpoR homo-dimerizes to activate JAK2, which in turn phosphorylates tyrosine residues on the receptor cytoplasmic tail permitting recruitment and phosphorylation of the signal transduction and activator of transcription (STAT)-5. Lenalidomide (LEN), a second generation immunomodulatory drug (IMiD), promotes the expansion of primitive erythroid progenitors in vitro and induces the expression of genes involved in erythroid lineage commitment (JCI 2008;118(1):248-58; PLoS medicine 2008;5(2):e35). In del(5q) clones, LEN is cytotoxic by inhibiting the haplodeficient serine/threonine phosphatase PP2A (Wei S, et. al PNAS 2009). To investigate the mechanism by which LEN promotes erythropoiesis, we investigated its action on EpoR cellular dynamics. Using the human erythroleukemia cell line, UT-7, treatment with LEN elicited concentration dependent upregulation of EpoR in whole cell lysates demonstrable by immunoblot (IB) analysis at concentrations ranging from 0.1–10μM, reaching a 2-fold increase vs. controls. Treatment with pomalidomide, an IMiD with superior erythropoietic activity, displayed greater potency, increasing cellular EpoR at concentrations as low as 1nM; whereas thalidomide was ineffective. Upregulation of EpoR was apparent as early as 1 hour (h) after LEN exposure, reaching maximal receptor induction at 8h. Isolation of membrane and cystosolic fractions followed by IB showed that EpoR was upregulated predominantly in the membrane fraction, increasing 4.3-fold. IB of detergent insoluble membrane raft (MR) fractions showed that LEN promoted lipid raft assembly and F-actin polymerization accompanied by translocation of EpoR into MR microdomains with JAK2, STAT-5 and Lyn signal intermediates. Pretreatment with the Rho-GTPase inhibitor, Y27632, suppressed LEN effects on MR and F-actin. Immunoprecipitation (IP) of EpoR showed that JAK2 co-precipitated with the receptor in LEN treated cells, suggesting drug induced release of pre-assembled EpoR/JAK2 complexes from golgi and endosomal compartments. Q-PCR showed no changes in EpoR gene mRNA expression up to 24 hours after drug exposure, supporting post translational drug effects. To confirm that the observed changes in EpoR dynamics in UT-7 cells extends to normal erythroid progenitors, we assessed EpoR expression and MR assembly (cholera toxin B labeled GM1) by fluorescence microscopy in CD71+ erythroid precursors from three normal bone marrow donors. Expression of EpoR increased a mean of 17% (range:0-68%) after 1h LEN [1μM] exposure (p=0.003), accompanied by an increase in MR aggregates. Because the LEN inhibitable target PP2A is a key regulator of Rho-GTPase guided receptor trafficking and AP-1 directed formation of MR targeted clathrin-coated vesicles, we examined its effects on EpoR induction. Pretreatment of UT-7 cells with the PP2A activator FTY720 antagonized the upregulation of EpoR by LEN, whereas treatment with the PP2A inhibitor cantharadin [0.5μM] induced EpoR protein expression. Inhibition of dynamin activity, a PP2A sensitive GTPase involved in type 1 receptor recycling from membrane to endosomes, recapitulated the effects of cantharadin, inducing EpoR expression. In summary, these findings indicate that LEN modulates EpoR equilibrium, mediated in part through its inhibitory effect on PP2A. LEN induces rapid translocation of mature EpoR/JAK2 complexes to the plasma membrane while promoting the assembly of MRs with signaling competent receptor platforms to optimize cytokine signal integrity. Disclosures:List:Celgene: Consultancy.

Epor Is Not Expressed or Functional in Primary Tumor Cells Isolated Directly From Human Primary and Metastatic Epithelial Tumors Including Breast, Non-Small Cell Lung, Colorectal, and Ovarian Tissues

Abstract 4781Several clinical trials have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents (ESA) in patients with cancer. One hypothesis proposes that ESAs stimulate tumor proliferation and or survival directly through binding and activating cell-surface receptors (EpoR). Expression and function of cell surface EpoR in human tumor tissue is controversial and has not been extensively investigated using validated reagents. MethodsEpoR expression and function was analysed in disaggregated primary tumor cells obtained from surgically resected human tumor tissues from 186 patients: colorectal (n=46), breast (n=34, including 8 Her2+ tumors defined by HercepTest™), non small cell lung (n=41), ovarian (n=35), Head and Neck (n=5) and other tumor types (n=8). Metastatic tissues were also analysed: colon (n=2), ovary (n=3), lung (n=2), pancreatic (n=1) as well as tumors from pre-treated patients: colorectal (oxaliplatin, bevacizumab; n=1), breast (cyclophosphamide, methotrexate, fluorouracil; n=2), breast (radiation, docetaxel cyclophosphamide, Paclitaxel; n=1), non small cell lung (radiation; n=2), ovarian (carboplatin and Paclitaxel; n=2).To examine if cell surface EpoR is expressed in the tumor cell compartment, flow cytometry analysis was carried out using a validated, EpoR-specific antibody (mAb 307). To evaluate if tumor cells respond functionally to recombinant human Epo (rHuEpo), levels of phosphorylated downstream signaling proteins (Akt, Erk1/2 and STAT5) were analysed by intracellular flow cytometry using phospho-specific antibodies following ex vivo stimulation with a titration of rHuEpo (0U [vehicle] to 300U/mL for 5 and 30 min). Expression of known tumor growth factor receptors (IGF-1R, c-Met and EGFR) was analyzed as positive controls. Additionally, the response of Akt, Erk and STAT pathways to stimulation with a tumor growth factor cocktail (“GF”: EGF, HGF and IGF-1), was used as a control for pathway integrity. The sensitivity of all flow based assays was validated through the analysis of EpoR expression and function in erythroid precursor cells (EPCs) across a time course of differentiation from primary human bone marrow samples. ResultsThough tumor samples exhibited cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface EpoR was detected in tumor cells from any of the 186 tumors examined by flow cytometry. Mean expression relative to negative control: EGFR = 2.80 (95% CI, 1.63–3.97), c-Met = 5.28 (95% CI, 4.39–6.17), IGF-1R = 8.03 (95% CI, 6.81–9.24) and EpoR (0.69 (95% CI, 0.64–0.73). No EpoR was detected by Western blot analysis of tumor lysates. As shown in Table 1, treatment with the GF cocktail stimulated phosphorylation of Akt and Erk in all cohorts. However, no activation of these pathways (or STAT5) by rHuEpo was observed in any of tumors even at high supra-pharmacological concentrations of rHuEpo (up to 300U/mL) and extended stimulation times (up to 30 mins).Table 1Mean response of signaling proteins to treatment with tumor growth factor cocktail (“GF”) and rHuEpo (300U/mL treatment shown). All values shown relative to vehicle controlPatient cohortpAktpErkGFrHuEpoGFrHuEpoBreast2.491.001.310.99Colon1.641.001.340.97Lung1.860.991.520.99Ovarian2.101.001.770.98Other2.071.021.340.94Pre-treated1.721.011.190.95Metastatic3.160.971.651.00Analysis of EpoR expression and function during differentiation in-vitro of EPCs revealed detectable EpoR cell surface expression (2.13-fold above baseline) and function (e.g. pSTAT5: 1.39-fold relative to vehicle at 300 U/mL rHuEpo) as early as day 1 when EPCs (CD36+/CD34-) comprised only 6.9% of the population. By day 8, (>90% EPCs) EpoR expression was detectable at 6.36-fold relative to baseline and pSTAT5 activation was observed 5.6-fold relative to vehicle at 300U/mL rHuEpo. ConclusionUsing sensitive methods no evidence of EpoR expression or function in primary human tumor cells isolated directly from primary and metastatic tumors was observed in multiple epithelial tumor types. Expression and functional activation of EGFR, c-Met and IGF1R was readily detectable. Therefore, this study suggests that it is unlikely that rHuEpo acts as a growth factor for primary human tumor cells. Disclosures:McCaffery:Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Elliott:Amgen Inc: Employment. Fitzpatrick:Amgen Inc.: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership.

Metabolism-Induced Reactive Oxygen Species and Hif1α-Mediated Gene Regulation Control the Timing and Magnitude of Hematopoietic Stem Cell Induction

Abstract 1266During development, the embryo is exposed to rapidly changing metabolic conditions and demands. The impact of elevated glucose levels on the developing hematopoietic system is not well characterized. Intriguingly, children born to mothers with gestational diabetes as well as those diagnosed with diabetes themselves have a higher risk of developing childhood leukemia, suggesting increased blood sugar concentrations may have lasting impact on hematopoiesis. To evaluate the consequences of elevated glucose levels on hematopoietic stem cell (HSC) development, zebrafish embryos were exposed to glucose and modifiers of glucose metabolism during embryonic blood cell specification from 12 to 36 hours post fertilization (hpf). HSCs in the Aorta-Gonad-Mesonephros (AGM) region were analyzed by in situ hybridization for the conserved markers runx1 and cmyb (n≥25–50 embryos/condition). D-glucose expanded HSCs in a dose-responsive manner, while the metabolically inactive enantiomer L-glucose did not affect HSCs. Quantitation by qPCR and FACS analysis of fluorescent HSC reporter embryos runx1:eGFP, cmyb:eGFP and CD41:GFP revealed a ∼3-fold increase in HSCs. Enhanced cellular proliferation (BrdU) was detected in the AGM in response to increased glucose levels. The effects of glucose on HSCs were sustained without any block in differentiation potential in larvae, and in adults after marrow injury. AGM time course analysis and ultrastructural assessment by electron microscopy (EM) revealed accelerated runx1 expression and hematopoietic cluster formation. EM and in situ hybridization for scl, gata1, and globin also demonstrated enhanced red cell numbers in response to elevated glucose levels. Genetic and chemical modulations of the metabolic hormone insulin did not alter the effects of glucose on HSC number. Activity of the glucose transporter glut1 was required to observe enhanced HSC induction, and glucose exposure increased glucose, ATP and lactate concentrations in the embryo. Additionally, incubation with inhibitors of glycolysis (lonidamine, ethyl-3-bromopyruvate) and oxidative phosphorylation (cyanide, oxaloacetate) reversed the beneficial effects of glucose, demonstrating that glucose affects HSCs specifically through energy metabolism. Enhanced oxidative phosphorylation produces excess reactive oxygen species (ROS), which can directly serve as hematopoietic signaling factors; treatment with the antioxidants N-acetylcysteine and MitoQ decreased HSC formation and blocked the effect of concomitant glucose exposure, while H2O2 expanded HSCs. Similarly, genetic enhancement of ROS levels by knockdown of peroxiredoxin 1 increased HSCs. ROS were visualized in vivo in erythrocytes and CD41+ cells using the fluorescent sensor peroxyfluorescein 2; increased glucose levels significantly enhanced ROS in the AGM as determined by FACS analysis of the lmo2:dsRed hematopoietic precursor and vascular reporter. ROS directly affects the stability of the cellular hypoxia sensor, hypoxia inducible factor 1α (hif1α), which can regulate expression of hematopoietic genes, such as vegf and epo. Stabilization of hif1α by cobalt chloride or morpholino knockdown of vhl enhanced HSCs and rescued the block in HSC induction following inhibition of energy metabolism or ROS production. Furthermore, by microarray and qPCR gene expression analysis, glucose exposure greatly impacted hematopoietic related transcripts, in addition to eliciting significant changes in the hif1α gene network: glucose increased expression of epo and epor during the primitive wave of hematopoiesis, confirming the effects on erythrocytes seen in vivo. In addition to vegf, nos and igf were induced during the definitive wave of hematopoiesis in a dose- and time-dependent fashion, providing signals to support HSC induction and expansion. These data indicate that fluctuations in glucose levels, and subsequent metabolic activity initially occurring in the dorsal aorta, are sensed by hif1α, which then functions to genetically enhance hematopoietic supply in advance of the anticipated demands of the growing organism during development. Our work provides direct evidence that the developing embryo responds dynamically to metabolic challenges by accelerating and expanding blood formation and demonstrates a connection between glucose metabolism and hif1α signaling in regulating HSC induction. Disclosures:Vander Heiden:Agios Pharmaceuticals: Consultancy, Equity Ownership. Goessling:Fate Therapeutics: Consultancy. North:Fate Therapeutics: Consultancy.

Absence of Functional Epo Receptors In Normal and Cancerous Human Tissues and Cell Lines,

Abstract 3169Erythropoietin (Epo) is a late acting growth factor, named because early studies suggested it had a singular effect on stimulation of red blood cell formation (erythropoiesis). Epo functions by activation of Epo receptors (EpoR) and while early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment, transcripts were later detected in other tissues and cell types including tumor cells with more sensitive RT-PCR strategies. This suggested that activation of EpoR by ESAs on tumor cells may explain results from some clinical studies where cancer patients in the ESAs treatment arm had decreased locoregional control and/or decreased survival. While EpoR transcripts could be detected, it was not clear that they were translated into EpoR protein or that the EpoR protein was functional. Attempts to examine EpoR protein expression in tumors were inhibited by nonspecificity and low sensitivity of available anti-EpoR antibodies.Recently specific and sensitive monoclonal antibodies were identified to human EpoR enabling experiments to explore EpoR protein expression in cells and tumors by western: CD34+ cells differentiated into erythroid progenitors and Epo-dependent UT-7/Epo cells served as positive controls and 769-P cells as a negative control. The latter expressed minimal EpoR mRNA and undetectable EpoR protein according to [125I]rHuEpo binding and western analysis. The limit of detection of EpoR by the most sensitive antibody, A82 in westerns was estimated to be less than 100 total (intracellular plus extracellular) EpoR/cell. By comparison, positive control cell types expressed over 10,000 EpoR/cell. In positive controls a 59 kDa protein, shown to be full-length EpoR, was readily detected with A82 with smaller EpoR proteins also present, but no proteins were detected in 769-P cells. The smaller proteins detected by A82 in the positive controls were shown by Mass-Spec analysis to be EpoR fragments. In contrast to the positive controls, EpoR proteins were not detected in either normal human breast or cancerous breast tissue (both Her2+ and Her2−) and there was little/no EpoR detected in a breast cancer lines (N=17) including MCF-7, though a band similar to EpoR in size was detected with the Santa Cruz anti-EpoR polyclonal, anti-peptide antibody, M-20. We therefore attempted to characterize this band. The band was not detected in MCF-7 cells with another specific anti-EpoR antibody (ab10653) nor was it observed in any immunoprecipitation:immunoblotting (IP:IB) experiments with combinations of 5 different anti-EpoR antibodies, though EpoR was readily detected in positive control. In contrast, the band was detected in IP:IB experiments with M-20 suggesting the protein is likely a non-EpoR protein that cross-reacted solely with that polyclonal antibody. Similar to breast tissues, EpoR was not detected with A82 in normal human primary keritinocytes, melanocytes and normal skin tissue. It was also not detected in squamous skin carcinoma and melanoma tissue or in cell lines derived from skin tumors (N=5). Matched normal and cancerous tissues from lung, colon, larynx, tongue and ovary were also negative for EpoR with A82. In contrast to tissues, low-level EpoR protein was detected in several of 21 cell lines derived from lung tumors. The EpoR protein in the one with the highest level (H838) was estimated to be ∼1,000–2,000 EpoR/cell, 10-fold lower than in erythroid progenitor cells and 40-fold lower than in UT-7/Epo cells. We therefore examined EpoR signaling in these cells. No phosphorylation of AKT or STAT5 (pAKT, pSTAT5) was detected in H838 cells with rHuEpo treatment. In control experiments increased pAKT was observed in H838 with a growth factor cocktail (EGF/ HGF/ IGF-1) and in UT-7/Epo cells treated with rHuEpo. These results suggested the EpoR in H838 cells was not functional. A factor dependent (IL-3) cell line (32D) was also engineered to express similar levels of EpoR as H838: these cells grew with IL-3 (10 ng/ml) but not with medium lacking growth factors, nor in medium containing 10 U/ml rHuEpo. In contrast, 32D cells with 20-fold higher levels of EpoR grew immediately with rHuEpo and at a rate similar to that with IL-3 but did not grow in absence of stimulation. These results suggest that EpoR expression level is important for Epo dependent growth of 32D cells, and that insufficient levels of EpoR in H838 cells may be one explanation for non-response to rHuEpo. Disclosures:Elliott:Amgen Inc: Employment, Equity Ownership. Swift:Amgen Inc: Employment, Equity Ownership. Busse:Amgen, Inc: Employment, Equity Ownership, Stock and Options. Rossi:Amgen: Employment, Equity Ownership. McCaffery:Amgen Inc: Employment, Equity Ownership.

Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production

While erythropoietin is the cytokine known that regulates erythropoiesis, erythropoietin receptor (EpoR) expression and associated activity beyond hematopoietic tissue remain uncertain. Here we show that mice with EpoR expression restricted to hematopoietic tissues (Tg) develop obesity and insulin resistance. Tg-mice exhibit a decrease in energy expenditure and an increase in white fat mass and adipocyte number. Conversely, erythropoietin treatment of wild-type mice increases energy expenditure and reduces food intake and fat mass accumulation but showed no effect in body weight of Tg-mice. EpoR is expressed at a high level in white adipose tissue and in the proopiomelanocortin neurons of the hypothalamus. While Epo treatment in wild-type mice induces the expression of the polypeptide hormone precursor gene, proopiomelanocortin, mice lacking EpoR show reduced levels of proopiomelanocortin in the hypothalamus. This study provides the first evidence that mice lacking EpoR in nonhematopoietic tissue become obese and insulin resistant with loss of erythropoietin regulation of energy homeostasis.

Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes

Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms of EpoR regulation in cardiomyocytes are unknown. We identified a 774 bp regulatory domain in the EpoR 5′ flanking region by reporter gene assays in murine HL-1 cardiomyocytes. The binding sites for Gata and Sp transcription factors both significantly contributed to EpoR promoter activity. DNA-binding studies (EMSA and ChIP assays) identified Gata4 and Sp1 as EpoR promoter-binding proteins in HL1 cardiomyocytes. Although Sp1 alone stimulates EpoR only slightly, forced expression of Gata4 significantly induced EpoR mRNA expression. In addition, knockdown of Gata4 (but also of Sp1) resulted in a significant decrease of EpoR transcript levels in HL-1 cardiomyocytes. Cumulative in vitro data suggest that function of the Sp1 site is essential for the Gata4-mediated transcription. In vivo, analysis of transgenic mice expressing an inducible small-hairpin RNA against Gata4 confirmed suppression of EpoR expression in the heart. Treating mice with high-dose doxorubicin not only resulted in Gata4 protein depletion, but also down-regulated EpoR, followed by up-regulation of EpoR transcripts when Gata4 levels recovered. In conclusion, we identified Gata4 as novel regulator of EpoR transcription in cardiomyocytes. In models of cardiac injury, down-regulation of Gata4 or Sp1 may limit the accessibility of the EpoR for binding of erythropoiesis-stimulating agents (ESA). Thereby our data underline the essential role of Gata4 in mediating cardioprotective effects.