Upregulation Of EpoR Research Articles

Therapeutic effects and related mechanisms of erythropoietin sustained-release gelatin hydrogel microspheres on a murine model of hindlimb ischemia

To investigate the therapeutic effects of erythropoietin sustained-release gelatin hydrogel microspheres (EPO-GHM) on a murine model of hindlimb ischemia and related mechanisms. Fifty two ten weeks old male C57BL/6J mice were assigned to 5 groups: sham-operated group (the right femoral artery suture was passed through the right femoral artery but not tied, n=8); saline group (right femoral artery ligation and intramuscular injection of saline at a dose of 4 ml/kg into the right hind limb, n=12); EPO group(right femoral artery ligation and intramuscular injection of EPO at a dose of 5 000 IU/kg into the right hind limb, n=12), empty GHM group (right femoral artery ligation and intramuscular injection of empty GHM at a dose of 4 ml/kg into the right hind limb, n=8); EPO-GHM group(right femoral artery ligation and intramuscular injection of EPO-GHM at a dose of 5 000 IU/kg into the right hind limb, n=12). The blood flow ratio of ischemic limb (right)/nonischemic limb (left) was measured using a laser Doppler perfusion imager. After 8 weeks, immunohistochemical analysis were used to evaluate the vessel density (vessel density of CD31 positive), arteriole density(vessel density of α-smooth muscle actin(α-SMA) positive) and muscle area(HHF35 positive area). The proliferating index of vessels was evaluated by double immunofluorescent labeling to evaluate effect of EPO-GHM on angiogenesis of ischemia limb. Western blot was used to evaluate the protein expression of EPO receptor, protein kinase B(AKT), p-AKT, endothelial nitric oxide synthase(eNOS), p-eNOS and matrix metalloproteinase 2(MMP-2). (1) Eight weeks later, the blood flow ratio of ischemic limb/nonischemic limb was significantly higher in the EPO-GHM group compared with other groups(0.810±0.080, 0.563±0.051, 0.570±0.056 and 0.561±0.052 respectively, all P<0.05). (2) CD31 antibody positive and α-SAM antibody positive densities were higher in the EPO-GHM group compared with other groups(P<0.01 or 0.05). (3)HHF35 positive area in saline group, EPO group, empty GHM group and EPO-GHM group were smaller than that of sham-operated group(all P<0.05). HHF35 positive area in saline group, EPO group, empty GHM group and EPO-GHM group were similar(all P>0.05). (4)The proliferating index of vessels was higher in the EPO-GHM group compared with other groups(P<0.01 or 0.05). (5) Compared with other groups, the protein levels of EPO receptor, AKT, p-AKT, p-eNOS and MMP-2 were significantly higher in EPO-GHM group(P<0.01 or 0.05) and level of eNOS was similar among five groups(P>0.05). RESULTS from present study suggest EPO-GHM could improve blood perfusion of ischemia limb in mice through increasing capillary and arteriolar densities and these beneficial effects are possibly mediated by EPOR up-regulation and AKT/p-eNOS/MMP-2 signaling pathway activation.

Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.

Nanoparticle facilitated inhalational delivery of erythropoietin receptor cDNA protects against hyperoxic lung injury

Our goals were to develop and establish nanoparticle (NP)-facilitated inhalational gene delivery, and to validate its biomedical application by testing the hypothesis that targeted upregulation of pulmonary erythropoietin receptor (EpoR) expression protects against lung injury. Poly-lactic-co-glycolic acid (PLGA) NPs encapsulating various tracers were characterized and nebulizated into rat lungs. Widespread NP uptake and distribution within alveolar cells were visualized by magnetic resonance imaging, and fluorescent and electron microscopy. Inhalation of nebulized NPs bearing EpoR cDNA upregulated pulmonary EpoR expression and downstream signal transduction (ERK1/2 and STAT5 phosphorylation) in rats for up to 21days, and attenuated hyperoxia-induced damage in lung tissue based on apoptosis, oxidative damage of DNA, protein and lipid, tissue edema, and alveolar morphology compared to vector-treated control animals. These results establish the feasibility and therapeutic efficacy of NP-facilitated cDNA delivery to the lung, and demonstrate that targeted pulmonary EpoR upregulation mitigates acute oxidative lung damage. From the Clinical EditorAcute lung injury often results in significant morbidity and mortality, and current therapeutic modalities have proven to be ineffective. In this article, the authors developed nanocarrier based gene therapy in an attempt to upregulate the expression of pulmonary erythropoietin receptor in an animal model. Inhalation delivery resulted in reduction of lung damage.

Mutation Of The Divalent Metal Transporter (Dmt1) Gene Results In Inefficient Induction Of The Erythroid Transcriptional Program Due To Latter Onset Of GATA-1 and Epor Expression

IntroductionDivalent metal transporter 1 (DMT1; SLC11A2) encodes trans-membrane protein variants that execute either non-heme iron absorption through apical duodenal membrane of enterocytes or transferrin-bound iron uptake through endosomal membrane of erythroid (and other) cells. Since 2005 several DMT1 mutations affecting accurate protein folding and localization were identified in patients with defective iron uptake leading to microcytic anemia, abnormal growth of erythroid progenitors in vitro, and iron overload in the liver. We have previously demonstrated that defective growth of DMT-1-mutatnt BFU-Es in vitro and anemia associated with ineffective erythropoiesis in vivo can be improved with high-dose erythropoietin (EPO) supplementation (Horvathova et al., 2012). Data from Dmt1-mutant mk/mk mice (Gunshin et al., 2005) suggested that the anemic phenotype is a result of ineffective erythropoiesis within bone marrow and spleen. HypothesisDMT1-mutant erythropoiesis inhibits EPO receptor (EPOR) signaling whose end-point target as well as upstream activator is the key transcription factor GATA-1. This results in defective erythroid development characterized by impaired survival capacity of erythroid progenitors, increased apoptosis of erythroblasts, and dysregulation of erythroid gene expression leading to ineffective erythropoiesis. MaterialsBone marrow cells of murine mk/mk mice were sorted using flow cytometry to obtain differentiating erythroid fractions based on antigens Cd71 and Ter119 (Sokolovsky et al., 2001) which were then subjected to gene expression analysis. ResultsFirstly, four consecutive fractions (proerythroblasts and early basophilic erythroblasts - stage I, late basophilic erythroblasts – stage II, chromathophillic and orthochromathophillic erythroblasts – stage III, and late chromathophillic erythroblasts and reticulocytes – stage IV) were isolated from mk/mk mice and control littermates. Flow cytometry showed enrichment in stages I and II and depletion in stage IV in the mk/mk bone marrow when compared to wild type controls, consistent with our previous data. In the spleen the major cellular enrichment was seen in stage III accompanied by cell depletion in stage IV. Gene expression of GATA-1 was markedly decreased at the onset (in stages I and II) of erythropoiesis while it was increased in terminal stage IV. The expression pattern of the GATA-1 target gene Epor was similar to that of GATA-1 expression, while expression of b-maj globin was significantly reduced indicating developmental delay of the erythroid Dmt1-mutant compartment (compared to wild type). Whereas GATA-1 and Epor expression is low in early erythropoiesis, a compensatory increase in their expression at later stages is not capable to efficiently upregulate b-maj globin. These data together with flow cytometry analysis identify a developmental blockade of erythropoiesis between stages II(III) and IV. To better understand whether Dmt1 levels regulate GATA-1 and Epor expression we used murine erythroleukemia (MEL) cells containing conditional (estrogen-regulated) transgene encoding GATA-1 fused with estrogen receptor ligand-binding domain (GER). MEL cells are cytologically characterized as proerythroblasts and early basophilic erythroblasts, stage I. Firstly, we established that GATA-1 upregulates Epor expression and directly binds to the Epor gene using RT-PCR, Immunoblotting, and chromatin immunoprecipitation (ChIP) in activated GER cells. Using ChIP-sequencing analysis of GATA-1 (and a panel of histone modifications) the GATA-1 enrichment was clearly identified at three distinct Epor regions in murine erythroblasts and differentiating MEL cells. Next, we downregulated Dmt1 using siRNA and observed that GATA-1-mediated upregulation of Epor in activated GER cells became inhibited. In addition, the knockdown of Dmt1 also inhibited steady state levels of GATA-1 in MEL cells by 25%. ConclusionsIneffective erythropoiesis in Dmt1-mutant mice is blocked at stages II (III) and display deregulation of the Epor signaling cascade involving GATA-1 and its targets. Our data thus interconnect iron uptake and the Epor/GATA-1 pathways and suggest their roles during erythroid pathogenesis upon DMT1 mutations. Grants: P305/11/1745, P301/12/P380, P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, GAUK 251135 82210 Disclosures:No relevant conflicts of interest to declare.

Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia

Managing critical limb ischemia (CLI) is challenging. Furthermore, ischemic myopathy prevents good functional outcome after revascularization. Hence, we have focused on limiting the tissue damage rather than angiogenesis, which has traditionally been the motivation to develop nonsurgical treatments for CLI. Erythropoietin (EPO) protects ischemic tissue, and this property may also benefit CLI. The objective of this study was to examine the expression of the tissue-protective EPO receptor complex (EPOR-CD131 [β-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor]) in skeletal muscle obtained from humans with CLI. Because native EPO is thrombogenic, the antiapoptotic and anti-inflammatory effects of a nonhematopoietic helix-B peptide of EPO (ARA 290) were investigated on ischemic myotubes invitro. Tissue was obtained from gastrocnemius muscle of 12 patients undergoing amputation for CLI and from 12 patients without limb ischemia. The expression of EPOR and CD131 was demonstrated by immunohistochemistry and Western blot. A validated invitro model of myotube ischemia was used in which mature C2C12 myotubes were cultured 6 to 12hours in a depleted media and gas mixture (20% CO2 and 80% N2). The myotubes were pretreated with EPO or ARA 290 before exposure to simulated ischemia. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase release assay. Enzyme-linked immunosorbent assay measured the inflammatory cytokines. EPOR and CD131 were expressed and significantly upregulated in CLI (average optical density [OD] in Western blot [control vs CLI] EPOR, 0.05 U vs 0.1 U; CD131, 0.10 U vs 0.22 U; P< .01). There was colocalization of EPOR and CD131 in the sarcolemma (cell membrane) of the skeletal myofiber. There was no difference in the distribution of colocalization between the CLI and the normal muscle. The ischemic myotubes treated by ARA 290 invitro had a significantly decreased number of apoptotic cells (ischemia vs ischemia plus ARA 290: 71.1% vs 55.1%; P< .01), cleaved caspase-3 (OD of ischemia vs ischemia plus ARA 290: 0.15 U vs 0.02 U; P< .01), lactate dehydrogenase release (ischemia vs ischemia plus ARA 290: 32.5 U/L vs 21.3 U/L; P< .01), and IL-6 release (OD at 450nm, ischemia vs ischemia plus ARA 290: 0.18 vs 0.13; P< .01). This study demonstrates the expression and the upregulation of EPOR and CD131 in CLI and also shows that EPOR and CDI are colocalized in the cell membrane of both ischemic and control muscle fiber. The invitro experiments demonstrate that ARA 290 decreases inflammation and apoptosis of ischemic myotubes. ARA 290 may potentially be used as adjunctive treatment for CLI.

Lenalidomide Upregulates Erythropoietin Receptor Expression Through Inhibition of the E3-Ubiquitin Ligase Ring Finger Protein 41 (RNF41)

AbstractAbstract 3455 Background:Lenalidomide (LEN) and its analogue, pomalidomide, promote erythroid lineage competence and in vitro colony-forming capacity. In patients with non-del(5q) myelodysplastic syndrome (MDS), LEN restores erythropoiesis in a subset of patients (List et al. N Eng J Med 2005;352:549). Investigations by Ebert et al. showed that such responders to LEN treatment display repression of erythroid specific genes and that LEN restored transcriptional response to erythropoietin (Epo) (Ebert BL et al. PLoS Medicine 2008;5(2):e35), suggesting that LEN enhances Epo receptor (R) signal fidelity. We previously reported that LEN induces cellular expression of JAK2 associated EpoR in a concentration-dependent manner, however, the mechanism of regulation is unclear (Basiorka et al. Blood 2011,118: 2382a). Recent investigations implicated inhibition of the cereblon RING (really interesting new gene) finger domain containing E3-ubiquitin ligase complex as a key target of the immunomodulatory drugs (IMiDs) responsible for the teratogenic effects of thalidomide and the cytotoxic effects of LEN in multiple myeloma (Ito T et al. Science 2010; 327:1345–50; Zhu YW et al. Blood 2011;118:4771–9). We recently showed that LEN also interacts with the RING finger E3 ubiquitin ligase, murine double minute 2 (MDM2) to inhibit ligase ubiquitination and stabilize the protein (Wei et al. Oncogene, MS#ONC-2011-01840R, 2012). Because EpoR turnover is regulated by ubiquitination and proteasomal degradation, we evaluated the effects of LEN on the E3-ubiquitin ligase, RNF41, which regulates steady state or ligand independent, Janus kinase (JAK2) associated Type I receptor internalization (Wauman et al. J Cell Science. 2011;124:921–932). We hypothesized that LEN upregulates JAK2/EpoR expression through inhibition of RNF41 function, thereby increasing EpoR expression and enhancing JAK2 competent receptor signaling. Methods and Results:Treatment of the UT-7 erythroid progenitor cell line with cycloheximide ±1μM LEN showed that LEN stabilized cellular EpoR (T1/2, LEN >72h vs. 56h). To determine if the effects of LEN on receptor turnover are restricted to Type 1 cytokine receptors, we examined the effects of LEN on cellular expression of IL3-R (Type 1) and c-Kit (Type 2). LEN up-regulated IL3-R expression in a concentration-dependent fashion, whereas c-Kit expression was unchanged, confirming Type 1 receptor specificity. To determine if LEN alters EpoR/RNF41 interaction, we assessed protein association after LEN treatment. Immunoprecipitation (IP) of either EpoR or RNF41 followed by immunoblot (IB) for the binding partner showed that LEN promoted EpoR/RNF41 association in a concentration dependent manner. To investigate the effects of LEN on RNF41 function, we assessed protein specific ubiquitination after proteasomal inhibition with bortezomib followed by LEN treatment. IP of RNF41 and EpoR followed by ubiquitin IB showed that LEN inhibited RNF41 auto-ubiquitination in a concentration-dependent fashion accompanied by a corresponding decrease in EpoR ubiquitination, suggesting that LEN inhibits RNF41 ubiquitination to increase EpoR accumulation. To confirm that RNF41 is the principal target of LEN responsible for EpoR stabilization, we transfected HEK293T cells with EpoR and/or RNF41 expression vectors using the calcium phosphate method. Steady state EpoR expression was lower in EpoR/RNF41 cells compared with cells transfected with EpoR alone. Moreover, EpoR upregulation by LEN was abrogated in EpoR/RNF41 cells indicating that cellular RNF41 is a critical determinant of EpoR upregulation by LEN. Immunohistochemical staining of 16 bone marrow biopsies from non-del(5q) LEN-treated MDS patients are in progress to determine the relationship between cellular RNF41 level in erythroid precursors and clinical response. Conclusion:Our findings suggest that LEN acts as a broad RING finger E3-ubiquitin ligase inhibitor, whose targets extend to the Type 1 cytokine receptor specific, RNF41. RNF41 inhibition by LEN promotes accumulation of signaling competent JAK2/EpoR complexes that may augment Epo responsiveness. Further investigation is warranted to determine if erythroid expression level of RNF41 may serve as a biomarker for response to LEN in patients with non-del(5q) MDS. Disclosures:List:Celgene: Consultancy.

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.

Hypoxia Induces Erythropoietin Receptor Expression on K562 Cell Line

ABSTRACTThe erythropoietin receptor (EpoR) and erythropoietin (Epo) mediate erythropoietin-induced erythroblast proliferation, differentiation and survival. This study examined the effects of the expression of EpoR on K562 (erythroleukemia) cells upon normoxia and hypoxia. In addition, the impact of the combined effect of recombinant human Epo and hypoxia was investigated. K562 (erythroleukemia) cells and as control group HL60 (promyeloblast) cells were cultured. Hypoxic incubation was performed with 5% O2, 5% CO2 and balance Nitrogen for 24 hours. After 24 hours, K562 and HL60 cells were transferred to normoxic and 5% hypoxic conditions. Recombinant Erythropoietin-alpha was added (10 U/ml) to the cells at the beginning of the experiment. Cultured cells were subjected to viability analysis, total RNA isolation, and protein isolation at three timepoints: 3 h, 6 h, and 24 h. Viability was analysed with a trypan blue exclusion using the Vi-Cell automated cell viability system. RT-PCR and western blot results of normoxic and hypoxic K562 and HL60 cell lines with/without rhEPO were compared.We showed that hypoxia upregulates the expression of EpoR on K562 erythroleukemia cells, and longer exposition to hypoxia turns the upregulated EpoR expression to basal levels. Recombinant human Epo (rhEpo) did not produce further impact in hypoxia-induced upregulation of EpoR neither in normoxia nor in hypoxia. Although addition of the rhEpo caused change in expression of cell-cycle regulators, it seems not to effect cell proliferation or cell viability. In the HL60 cell line, however, we detected EpoR mRNA, but not Epo mRNA by RT-PCR. Hypoxia did not alter the level of EpoR mRNA expression.The results of this study suggest that the expression of EpoR might be regulated by hypoxia, but not by Epo, and the expression of cell-cycle regulators might be regulated by both hypoxia and Epo.

Epor Over-Expression In TEL-AML1 Positive ALL: Critical Roles for DNA Methylation, GATA-2 or MicroRNAs?

AbstractAbstract 3619Erythropoietin (Epo) and its receptor (EpoR) play vital roles in regulating red blood cell production. However, aberrant over-expression of EpoR has been reported in a wide range of tumours and hematological malignancies, particularly acute lymphoblastic leukemia (ALL) with the chromosomal translocation TEL-AML1 (ETV6/RUNX1). Around 25% of B-cell ALLs contain this chromosomal translocation which is associated with a favourable, prognosis but the role of EpoR is unknown.We have investigated whether methylation, transcription factors or miRNA regulation are implicated in the mechanism of the specific EpoR up-regulation associated with the TEL-AML1 rearrangement using two human B precursor cell line models. The pre-B REH cell line is TEL-AML1 positive whereas the pre-B NALM-6 cell line is TEL-AML1 negative; in addition the erythroleukemia cell line UT-7, which constitutively over- expresses EpoR was used as a positive control. Sequencing confirmed that the EpoR in these cell lines was wild type. Quantitative RT-PCR (RQ-PCR) of EpoR mRNA levels showed ~25 fold higher expression in REH cells compared to NALM-6 cells, verified by western blot analysis. RQ-PCR analysis of 20 pediatric ALL patient samples confirmed a 14-fold up-regulation (p<0.05) of EpoR in TEL-AML1 positive patients compared to hyper-diploid ALL patients without the translocation. Furthermore, the EpoR is functional in REH cells as serum starvation followed by stimulation with 10 U/ml Epo clearly activated the JAK2/STAT5, PI3K/Akt and Ras/ERK downstream signaling pathways within 15 min. In contrast, NALM-6 cells showed no activation of the signaling pathways in response to Epo.DNA methylation is an epigenetic marker which can directly impact on gene transcription, with aberrant methylation reported in many cancers. The methylation status of 18 CpG sites over 342 bp within the EpoR promoter encompassing the GATA and Sp1 binding sites was analysed by pyrosequencing in REH and NALM-6 cells. Fifteen of the 18 CpG sites showed significantly higher (p<0.001) levels of methylation in the NALM-6 cells (13-71%) compared to REH (2.4-10%) and UT-7 (1.8-8.5%) cells. The 15 significantly different sites had an average of 4.1% methylation in REH and UT-7 cells and 36.6% methylation in NALM-6 cells, showing a difference of 32.5% between the REH and NALM-6 cells. Comparable levels of methylation were seen at three CpG sites (7, 8 and 18) located near the Sp1 binding site. Treatment of NALM-6 cells with the IC50 dose (0.5 μmol) of Decitabine resulted in a regional decrease in methylation but no overall increase in EpoR expression whereas treatment of REH cells with Decitabine had no effect on methylation or expression. This suggests that methylation status of the EpoR promoter is altered by the presence of the TEL- AML1 fusion protein but may not be the critical factor in the up-regulation of EpoR expression.The GATA family of zinc-finger proteins have well characterised roles in hematopoiesis and EpoR regulation. Measurement of the expression of all six GATA transcription factors, by RQ-PCR showed that only GATA-2 was significantly up-regulated in REH cells (p<0.001) compared to the TEL-AML1 negative NALM-6 cell line with a Ct difference of 8.6 (~388 fold change). The up-regulation of GATA-2 was recapitulated in 10 TEL-AML1 positive patients and 10 hyper-diploid ALL patients who showed an average 28-fold difference in expression (p<0.0001). An increase in GATA-2 protein levels was also evident in REH cells using western blot analysis.Furthermore, initial studies have indicated differential expression, between the REH and NALM-6 cell lines, in miRNA whose targets have been predicted (www.diana.pcbi.upenn.edu) to be EpoR and/or GATA-2. Further investigation of the modulation of EpoR expression by methylation, GATA-2 or miRNAs, will lead to a greater understanding of of the factors regulating the expression of EpoR and the potential for alternative forms of therapeutic intervention in patients with TEL-AML1 ALL. Disclosures:No relevant conflicts of interest to declare.

Gata4 Regulates Expression of the Erythropoietin Receptor in Cardiomyocytes.

Abstract 3610Poster Board III-546Experimental 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. Using reporter gene assays, we identified in murine HL-1 cardiomyocytes a 774 bp regulatory domain in the 5′-flanking region of the EpoR gene. Fragments that include an E-box, CACCC motifs, GC-rich elements, and binding sites for members of the Gata and Sp transcription factor families contribute to EpoR promoter activity. EMSA experiments indicated the specific binding of Gata4 and Sp1 (a known interaction factor of Gata4) to the minimal EpoR promoter. Forced expression of Gata4 resulted in a significant induction of EpoR mRNA expression, while Sp1 overexpression did not affect EpoR expression. Depletion of Gata4 by treating mice with high-dose doxorubicin led to the downregulation of EpoR expression, followed by the upregulation of EpoR when Gata4 levels recovered after 5 days. Transgenic mice with an inducible short hairpin RNA against Gata4 confirmed the suppression of EpoR expression if Gata4 levels were reduced. Under hypoxia, however, a 2-fold induction of EpoR mRNA expression in cardiomyocytes was independent from Gata4, since Gata4 levels decreased. Our data indicate that Gata4 activates EpoR mRNA expression in cardiomyocytes. This is a novel mechanism that differs from EpoR regulation in neurons and hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

Differential up-regulation of erythropoietin and its receptor in benign and malignant prostatic tissue

Purpose To investigate differential up-regulation of erythropoietin and its receptor in benign and malignant prostatic tissue. Materials and methods An immunohistochemical analysis of EPO and EPOR expression was performed on 30 cases of prostate carcinoma (PCa) with 16 high-grade prostate intraepithelial neoplasia (PIN), 50 cases of benign prostatic hyperplasia (BPH) lesions, and 30 normal prostatic tissue samples as control. Results Over-expression of EPOR was only shown in PCa and high-grade PIN tissue but over-expression of EPO was also shown in BPH tissue. There was significant relationship between EPO and EPOR score in BPH, high-grade PIN, and PCa but no association between EPOR or EPO score and Gleason score in PCa. Conclusions Up-regulation of EPOR has a more important role in comparison with EPO in prostate carcinogenesis. Differential over-expression of EPOR and EPO in benign and malignant prostatic tissue is ascribed to different mechanisms involved in up-regulation between EPO and EPOR.

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.

Erythroid expansion and survival in response to acute anemia stress: The role of EPO receptor, GATA‐1, Bcl‐xL and caspase‐3

Erythropoietic stress occurs under conditions of tissular hypoxia, such as anemia. Functional relationships between erythroid bone marrow (BM) proliferation, differentiation, the expression of survival and apoptotic related proteins, as well as the features of the BM microenvironment upon acute anemic stress, are not fully elucidated. To achieve this aim, CF-1 Swiss mice were injected with a single dose of 5-fluorouracil (5-FU, 150 mg/kg ip) and a multiparametric analysis was conducted for 20 days. Apoptosis (TUNEL assay), BM architecture organization (scanning electronic microscopy), proliferation (DNA assay), differentiation (clonogenic cultures), expression of survival erythroid related proteins (EPO-R, GATA-1, Bcl-xL) as well as the expression of apoptotic- related proteins (Bax, activated Caspase-3) by Western blotting, were evaluated. Experimental data showed that apoptosis, arrest of cell proliferation and disruptions of BM architecture were maximal within the first period of acute stress (1-3 days). Bax and caspase-3 overexpressions were also coincident during this acute period. Moreover, from day 5 upon drug challenge BM responds to acute stress through the EPO-EPO-R system, prompting expressions of GATA-1 and Bcl-xL. Erythroid proliferation rates and red-cell-committed progenitors enhanced in a coordinated way to restore the size and function of the red cell compartment. A second overexpression wave of active caspase-3 was noticed during stress recovery. Together, these results indicate that in response to acute stress a dramatic increase in CFU-E (erythroid colony forming units) population is concomitant with upregulation of EPO-R, GATA-1 and Bcl-xL in the BM erythroid compartment, and that these concurrent processes are crucial for acquiring proper erythroid cell functionality without delayed response to tissular hypoxia.

Role of erythropoietin receptor expression in malignant melanoma

9070 Background: Erythropoietin (Epo) is known to promote proliferation and differentiation of erythroid precursors and to prevent apoptosis. Epo receptor (EpoR) belongs to the cytokine receptor superfamily and exerts its effects via receptor homodimerization, subsequent phosphorylation of Janus kinase 2 (Jak2) and activation of signal transducers and activators of transcription (STAT)-3 and -5. Epo is also able to induce the activation of the Ras-Raf-MEK-ERK pathway in different types of cells. Recombinant human erythropoietin (rHuEpo) can prevent anemia in tumor patients; recent evidence has suggested that rHuEpo may adversely affect the survival of selected cancer patients by promoting tumor growth. Methods and Results: In this study, we have investigated the expression of EpoR in malignant melanoma specimens and analyzed the effect of rHuEpo on proliferation and apoptosis of melanoma cells in vitro. We observed a transcriptional upregulation of EpoR in 50% of malignant melanoma metastases in comparison to benign nevi and primary melanoma using quantitative real- time RT-PCR. EpoR expression was weak in normal human melanocytes (NHM) and different melanoma cell lines (e.g. A375 and BLM cells), but was high in MV3 cells and melanoma metastasis specimens as assessed by Western blot analysis. Furthermore, rHuEpo treatment also induced the phosphorylation of JAK-2, STAT-3 and activated the Ras-Raf-ERK pathway as shown by Western blot experiments. In addition, rHuEpo seemed to prevent the cisplatin-induced apoptosis in certain melanoma cell lines. Conclusions: In summary, we demonstrate functional EpoR expression in stage IV melanoma specimens, thus cautioning the use of rHuEpo for the treatment of anemia in these patients. No significant financial relationships to disclose.

Intravitreal Injection of Erythropoietin Protects both Retinal Vascular and Neuronal Cells in Early Diabetes

To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes. Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC). In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO. These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR.

Accelerated Proliferation and Limited Differentiation, Mediated through Jak2, Exacerbate Ineffective Erythropoiesis in β-Thalassemia.

Ineffective erythropoiesis (IE) in β-thalassemia has been attributed to erythroid cell death mediated by apoptosis or hemolysis during the maturation process. Historically, ferrokinetic studies in this disease suggested that 60%–80% of erythroid precursors die in the marrow or extramedullary sites. However, several observations have challenged this view. First, the number of apoptotic erythroid cells in patients is low compared to net expansion of the erythroid cell pool. Second, hemolytic markers in β-thalassemic patients are normal or only slightly increased, unless additional pathological conditions appear. Third, our most recent study (Blood, Gardenghi et al, 2007 Jun 1) demonstrated that GI iron absorption in β-thalassemia is increased by the dysregulation of genes such as hepcidin and ferroportin that control iron absorption, resulting in iron levels that exceed the amount required for erythropoiesis. We have undertaken a detailed investigation using cohorts of mice (n>30 per genotype) with β-thalassemia intermedia (th3/+) and major (th3/th3). Using these models, we have previously shown that the severity of anemia (as low as 1 g/dL) inversely correlates with the total number of nucleated erythroid cells (»100 fold compared to wild-type (wt) mice). Cytological analysis has clearly shown that thalassemic spleen specimens were comprised of a homogeneous pre-erythroblastic population. In contrast, the percentage of apoptotic cells and the level of hemolytic markers, such as bilirubin and lactic acid dehydrogenase, slightly increased or were not different compared to wt mice. While not excluding a role for apoptosis, our observations suggest that control of the cell cycle and maturation of erythroid precursors play an important role in IE. We then explored whether the erythroid cell cycle was dysregulated in our model system. We found that erythropoietin (Epo) levels were raised in thalassemic animals by as much as three orders of magnitude. Binding of Epo to its receptor (EpoR), activates antiapoptotic and cell cycle promoting genes, through activation of Jak2 and Stat5. By Western blot we demonstrated up-regulation of EpoR, Stat5 and the antiapoptotic protein BclXL, as well as that of proliferation promoting genes, such as CycA and Cdk2, in purified thalassemic erythroid cells compared to those of wt animals. This data was confirmed by staining both wt and thalassemic liver and spleen sections using the proliferation markers Ki67 and Mcm3, by clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after BrdU injection. In the latter case, we observed 22%, 30% and 44% BrdU+ cells from wt, th3/+ and th3/th3 mice, respectively. In addition, freshly purified thalassemic erythroid cells proliferate faster in vitro than normal cells, a phenomenon blocked by AG490, a Jak2 inhibitor. Significantly, we have been able to reproduce results from our animal studies in humans, comparing normal and thalassemic blood and spleen specimens. In conclusion, we propose that IE in β-thalassemia is likely to be the result of altered cell proliferation and impaired cell differentiation, which in turn limit apoptosis, thereby mimicking tumor-like behavior.

Brain erythropoietin receptor expression in Alzheimer disease and mild cognitive impairment.

Cellular mechanisms conferring neuroprotection in the brains of patients with Alzheimer disease (AD) remain incompletely understood. Erythropoietin (Epo) and the erythropoietin receptor (EpoR) are expressed in neural tissues and protect against oxidative and other stressors in various models of brain injury and disease. Our objective in this study was to determine whether EpoR is upregulated in the brains of persons with sporadic AD and mild cognitive impairment (MCI). Postmortem hippocampus and temporal cortex from subjects with AD, MCI, and no cognitive impairment (NCI) were procured from the Religious Orders Study. Total immunoreactive EpoR protein was determined by Western blotting. Astrocytes expressing immunoreactive EpoR were quantified in 4 temporal and 6 hippocampal regions, and correlated with clinical, neuropsychologic, and neuropathologic indices. Total immunoreactive EpoR protein was markedly increased in AD and MCI temporal cortex versus NCI tissues. Composite measures of glial EpoR expression in temporal cortex layers I to IV were significantly greater in the MCI group compared with the NCI and AD groups. Hippocampal EpoR scores were increased in persons with MCI and AD relative to those with NCI. There was substantial subregional heterogeneity in disease-related EpoR expression patterns in AD and MCI temporal cortex and hippocampus. There was no association of EpoR-positive astrocytes with summary measures of global cognition or AD pathology. We conclude that upregulation of EpoR in temporal cortical and hippocampal astrocytes is an early, potentially neuroprotective, event in the pathogenesis of sporadic AD.

Erythropoietin is neuroprotective on GABAergic neurons against kainic acid-excitotoxicity in the rat spinal cell cultures

The aim of this study was to investigate whether erythropoietin (EPO) protect the spinal GABAergic neurons against kainic acid (KA)-excitotoxic damage in rat spinal cord cell cultures. We performed immunohistochemical staining and Western blotting of glutamate decarboxylase 67 (GAD67), one isoform of GABA-producing enzyme, which was considered to have involved in nonsynaptic functions, such as energy metabolism or trophic support. T exposure to KA significantly reduced the intensity of GAD67 expression in the GABAergic neurons and whole cell lysate, indicating that the excitotoxic damage on the GABAergic neurons may lead to reduction of the GAD67 production following KA-exposure. We found that post-treatment of EPO for 48 h after KA-injury remarkably enhanced the expression level of GAD67 and erythropoietin receptors (EpoR), which were deteriorated by KA. Our confocal images clearly demonstrated a remarkably enhanced expression of EpoR on the surface of the GABAergic neurons by post-treated EPO after KA-damage. This result suggests that the neuroprotective effect of post-treated EPO on the GABAergic neurons can be related to the EPO-mediated EpoR upregulation following KA-excitotoxicity. We observed that the post-applied EPO clearly increased expression of tyrosine Janus kinase 2 (JAK2), which is known to be the first step of EpoR-stimulation. In conclusion, the post-treated EPO is a potent protector of the spinal GABAergic neurons against KA-excitotoxicity and regulates production of GAD67 for the multiple trophic roles after KA-induced disturbance. We suggested that the protective effect of post-treated EPO on the GABAergic neurons is mediated by signal transduction involving EpoR-dependent JAK2 pathway.

Erythropoietin is neuroprotective against NMDA-receptor-mediated excitotoxic brain injury in newborn mice

Using an established mouse model of human periventricular leukomalacia, we investigated whether EPO could reduce excitotoxic damage. When administered 1 h following intracerebral injection of 10 microg ibotenic acid at day 5 of life, both a single injection of EPO (5000 IU/kg bw) and repetitive administrations of EPO reduced white and gray matter lesion size. The therapeutic window for protection was small as the protective effect of EPO was lost when EPO administration was delayed to 4 h post-insult. EPO-mediated upregulation of EPO-R, but not EPO, mRNA was observed within 4 h of the excitotoxic insult. The EPO effect was gender independent. Minor hematopoetic effects were observed following EPO treatment. We conclude that a single dose of EPO is sufficient to reduce excitotoxic brain injury and may therefore possess therapeutic relevance in the clinical setting.

A Novel Mechanism of Cooperation between c-Kit and Erythropoietin Receptor: STEM CELL FACTOR INDUCES THE EXPRESSION OF Stat5 AND ERYTHROPOIETIN RECEPTOR, RESULTING IN EFFICIENT PROLIFERATION AND SURVIVAL BY ERYTHROPOIETIN

Optimal production of red cells in vivo requires collaboration between c-Kit, erythropoietin receptor (Epo-R), and GATA-1. However, the mechanism(s) of collaboration remain unclear. Utilizing an embryonic stem cell-derived erythroid progenitor cell line from mice deficient in GATA-1, we have examined the role of c-Kit and Epo-R in erythroid cell proliferation, survival, and differentiation. In the absence of GATA-1, we demonstrate an essential role for c-Kit in survival and proliferation of erythroid progenitors via the regulation of Bcl-2 expression. In addition, we demonstrate that Epo-R and Stat5 are regulated by a second, novel mechanism. We demonstrate that c-Kit stimulation by stem cell factor is essential for the maintenance of Epo-R and Stat5 protein expression, which results in significantly enhanced Bcl-x(L) induction and survival of erythroid progenitors in response to Epo stimulation. Restoration of GATA-1 function results in terminal erythroid maturation and up-regulation of Epo-R and Bcl-x(L) expression, leading also to significantly enhanced survival of terminally differentiating erythroid progenitors in the presence of only Epo. These results demonstrate that c-Kit and Epo-R have unique role(s) during distinct phases of erythroid maturation, and both stem cell factor and Epo contribute to the regulation of the Epo-R-Stat5-Bcl-x(L) pathway to ensure optimal survival, proliferation, and differentiation of erythroid progenitors.