Erythropietin (Epo), acting through its receptor (EpoR), is an essential hemotopietic growth factor that regulates the proliferation, differentiation, and survival of erythroid progenitor cells. Perturbations of Epo/EpoR function cause myeloproliferative disease, such as erythrocytosis, or myelodeficient disease, such as anemia. Therefore, defining the mechanisms by which Epo/EpoR control proliferation and differentiation of erythroid cell lineages attracts interest. Ubiquitin-dependent internalization and degradation is a common regulatory mechanisms affecting signaling from a variety of receptors. Although EpoR has been found to be ubiquitinated, the function of EpoR ubiquitination in the regulation of Epo signaling remains unclear. Therefore, the primary goal of this study was to define the role of EpoR ubiquitination in regulating Epo signaling activities and erythroid cell growth. We showed that EpoR was ubiquitinated in response to ligand stimulation, and that loss of EpoR ubiquitination reduced signaling activity and biological responses to low dosages of Epo. We also identified two EpoR lysines that were the primary targets for ubiquitination, and showed that either ubiquitination site supported the enhanced activities of wild-type-EpoR. Ubiquitination of EpoR was also associated with a change in the endocytic pathway mediating internalization of EpoR. Specifically, constitutive internalization of non-ubiquitinated EpoR was found to depend on dynamin activity, while internalization of ubiquitinated EpoR was dynamin-independent but could be inhibited by disrupting lipid raft microdomains in the plasma membrane. Interestingly, inhibiting internalization of ubiquitinated EpoR (by disrupting lipid rafts) specifically reduced signaling from ubiquitinated receptors without affecting signaling from non-ubiquitinated receptors. Conversely, reducing internalization of non-ubiquitinated EpoR (by inhibiting dynamin) reduced its signaling activity without affecting signaling from ubiquitinated receptors. This strong correlation between EpoR internalization and signaling activity suggests a novel regulatory mechanism in which internalization of EpoR facilitates its signaling activity. In this regard, Epo-induced ubiquitination of EpoR promotes more efficient internalization of ligand-activated receptor and may contribute to enhanced responsiveness to low concentrations of Epo.

Yes / Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been contro- versial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that sev- eral of these specifically recognize EPOR by Western blot, immunoprecipi- tation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment. / Study was supported by the FP7-Health European commission EpoCan grant (282551).

In USA more than 12.5% of all infants are born preterm. Approximately 75% of all perinatal deaths occur among these preterm infants. Preterm infants are frequently very low in birth weight (VLBW) and receive multiple red blood cell (RBC) transfusions. These transfusions pose increased risk of infections and other complications. Since erythropoietin (EPO) stimulates RBC production, EPO treatment of VLBW infants has received attention as a modality for reducing transfusions in this group. The overall hypothesis of this work is that treatment optimization of EPO of anemia in preterm infants requires a comprehensive knowledge of the behavior of RBC and the pharmacokinetic/pharmacodynamics (PK/PD) relationship between EPO and erythropoiesis. Under that overall hypothesis, the specific aims were: 1) To describe erythropoiesis dynamics in preterm infants, 2) To determine and explain the variability in the response to EPO in preterm infants, 3) To evaluate newborn sheep as an experimental model for erythropoiesis in preterm infants, 4) To test the hypothesis that RBC lifespan is shortened under acute hypoxic stress conditions, 5) To test the hypothesis that EPO receptor (EPOR) pool size increases under hypoxic stress conditions and the change in EPOR pool size can be predicted using EPO clearance measurements, 6) To describe the effect of EPOR pool size changes on erythropoiesis kinetics. A model that describes erythropoiesis dynamics in preterm infants as a function of the plasma EPO concentration is presented in Chapter 2. In Chapter 3, several covariates are tested for their ability to identify infants with good EPO responsiveness. The lamb is also tested as an animal model for the erythropoiesis in preterm infants (Chapter 4). In Chapters 5-7, the effect of hypoxic stress conditions on RBC survival was explored defining the relation between the efficacy of EPO and survival of RBC produced as a result of EPO administration. RBC lifespan measurement methods are reviewed in Chapter 5. In Chapter 6, a new methodology for the measurement of RBC lifespan under stress conditions is developed. This new methodology is applied in Chapter 7 to explore the effect of hypoxic stress conditions on the survival of RBC. The study presented in Chapter 8 is undertaken to investigate changes in both EPOR pool size and EPO clearance under hypoxic conditions. An erythropoiesis model that accounts for change in the EPOR pool size under stress conditions is presented in Chapter 9. Analysis of erythropoiesis dynamics in preterm infants demonstrated that a three fold increase in the amount of RBC produced by preterm infants is possible by EPO administration. This emphasizes the potential of using EPO for the management of anemia in preterm infants. Covariate screening identified gestational age as a potential marker for the responsiveness to EPO treatment. PD analysis results in lambs demonstrated similarities between lambs and preterm infants in different erythropoietic characteristics such as sensitivity to EPO in producing RBC, Hb production rate before birth and blood volume. Survival analysis demonstrated that RBC lifespan is not shortened under acute hypoxic conditions Analysis of EPOR mRNA level demonstrated an up regulation of EPOR level under stress conditions accompanied by a parallel increase in EPO clearance. EPOR up regulation under stress conditions level was incorporated in a PD model presented in Chapter 9. The developed model provides a framework for optimizing EPO dosing. Accordingly, an optimal dosing strategy should in general maximize the interaction between EPO and EPOR. Specifically, EPO should be administered when the number of EPOR are close to maximally up-regulated.

Erythroid terminal differentiation couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. I used Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. I found that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. Specifically, I found that high levels of EpoR signaling increase the size and shorten the cycle of early erythroblasts, which are amongst the fastest cycling somatic cells. I confirmed the effect of erythropoietin (Epo) on red cell size in human volunteers, whose mean corpuscular volume (MCV) increases following Epo administration. Our work shows that EpoR signaling alters the expected inverse relationship between cell cycle length and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress. The ability of EpoR signaling to increase cell size in rapidly cycling early erythroblasts suggests that these cells have exceptionally efficient EpoR-driven mechanisms for growth. I found evidence for this in ongoing work, where Epor−/− and Stat5−/− single-cell transcriptomes show dysregulated expression of ribosomal proteins and rRNA transcription and processing genes. Global rates of ribosomal rRNA transcription and protein synthesis increase in an EpoR dependent manner during a narrow developmental window in early ETD, coincident with the time of cell cycle shortening. Our work therefore suggests EpoR-driven regulation of ribosome biogenesis and translation orchestrating rapid cycling and cell growth during early ETD.

Derzeit wird die Anwendung von Erythropoietin (Epo) zur Neuroprotektion in präklinischen und klinischen Studien intensiv untersucht. Für die Neuroprotektion ist die Regulation des Erythropoietin-Rezeptors (EpoR) in neuronalen Zellen von hoher Relevanz. In dieser Arbeit wurden die transkriptionellen Mechanismen der EpoR-Regulation in humanen Neuroblastom-Zellen SH-SY5Y mit neuronalem Phänotyp untersucht. Da der hämatopoietische Transkriptionsfaktor GATA-1 die EpoR-Transkription in erythroiden Vorläuferzellen in Kooperation mit Sp1 stimuliert, wurde die Rolle der in neuronalen Vorläuferzellen exprimierten GATA-Transkriptionsfaktoren bei der EpoR-Expression untersucht. Es wurde eine in vitro Bindung von GATA-2, -3 und -4 an zwei Motive in der EpoR 5’-flankierenden Region (-274/-271; -47/-44) nachgewiesen. In Reportergen-Assays zeigten diese Genabschnitte eine bis zu 4,8-fache Aktivitätssteigerung bei Überexpression von GATA-2, -3 oder -4. Die endogene EpoR mRNA-Expression wurde dadurch aber nicht erhöht. Hypoxie (2% O2, 3 d) erhöhte die EpoR-Expression signifikant (1,8-fach, p < 0,01), wobei überexprimierte GATA-Transkriptionsfaktoren diesen Effekt nicht weiter steigerten. Die Gabe von Epo (5 U/ml, 3 d) hatte weder unter Normoxie noch unter Hypoxie einen Einfluss auf die EpoR-Expression. Die Promotoraktivität der Reporterkonstrukte wurde durch Mutation der GATA-Bindungsstellen nicht verändert, jedoch bei mutierter Sp1-Bindungsstelle inhibiert. Ein Fragment der 5’-flankierenden Region (-449/-285), das ein Cluster von Bindungsstellen für unterschiedliche Transkriptionsfaktoren enthält, zeigte die stärkste Promotoraktivität und rekrutierte offenbar die RNA-Polymerase II. In unserem Modell spielen die GATA-Faktoren keine direkte Rolle für die EpoR-Genregulation in neuronalen Vorläuferzellen. Die EpoR mRNA-Expression wird eher durch einen Komplex aus verschiedenen Transkriptionsfaktoren reguliert, der an eine 5’ des minimalen Promotors liegende DNA-Region zu binden scheint. / Since the use of erythropoietin (Epo) as neuroprotective agent is currently intensively studied in preclinical and clinical trials, regulatory mechanisms of the erythropoietin receptor (EpoR) in neuronal cells are of particular interest. In this study, the transcriptional mechanisms of EpoR regulation were analyzed in human neuroblastoma-derived SH-SY5Y cells, which exhibit a neuronal phenotype. Considering that the hematopoietic transcription factor GATA-1 stimulates EpoR transcription in cooperation with Sp1 in erythroid progenitors, the role of other GATA family members expressed in neuronal precursor cells were studied. In vitro, GATA-2, -3 and -4 were found to bind to two consensus motifs within the EpoR 5’-flanking region (-274/-271 and -47/-44). In reporter gene assays, these regions showed an up to 4.8-fold induction if GATA-2, -3 or -4 were overexpressed. However, forced expression of GATA-2, -3 and -4 did not enhance endogenous EpoR mRNA expression. Under hypoxia (2% O2, 3 d), EpoR expression was significantly upregulated in SH-SY5Y cells (1.8-fold, p < 0.01), but not further increased by the additional overexpression of the GATA factors. Incubation of the SH-SY5Y cells with recombinant Epo (5 U/ml, 3 d) had no effect on the EpoR expression under normoxia or hypoxia. The promoter activities of the reporter constructs were not changed by mutations in the GATA sites, but abolished by mutations of Sp1 binding sites. A fragment (-449/-285) of the 5’-flanking region that contains a cluster of binding sites for various transcription factors showed strongest promoter activity and was obviously directing the recruitment of RNA polymerase II. We conclude that GATA factors do not play a major role in regulating EpoR expression in our model for neuronal precursor cells. EpoR mRNA expression is rather regulated by a complex of various transcription factors, which may bind to a region upstream of the minimal promoter.

No description available.

O estudo teve como objetivo avaliar a razão de custo-efetividade, sob a perspectiva do Sistema Único de Saúde SUS, do tratamento da anemia de pacientes em Terapia Renal Substitutiva. Duas alternativas foram comparadas: um novo medicamento recentemente registrado no Brasil, o Ativador Contínuo de Receptor de Eritropoetina (Continuous Erythropoietin Receptor Activator), CERA, e outro, atualmente disponível no sistema de saúde brasileiro, a Eritropoetina Recombinante Humana - Epo-rHu. Métodos: Um modelo de Markov simulou o curso de uma coorte de pacientes em Terapia Renal Substitutiva tratados com CERA e Epo-rHu por quatro anos. A qualidade de vida associada ao uso dos medicamentos foi estimada de forma indireta, por meio de entrevista qualificada com os profissionais cuidadores, previamente submetida e aprovada pelo Comitê de Ética em Pesquisa local. Foi realizada análise de sensibilidade no modelo proposto através da variação dos parâmetros: dose dos medicamentos, custo das estratégias, taxa de desconto e efetividade utilizados para sua construção. Resultados: A média da qualidade de vida atribuída aos pacientes tratados foi 6,3 para a Epo-rHu, 7,8 para o CERA e 9,3 para os pacientes transplantados. O modelo demonstrou que a estratégia mais custo-efetiva é a terapêutica com a Epo-rHu, com um custo por QALY de R$ 21.052,00. O custo incremental por QALY ganho associado ao CERA foi de R$ 72.974,00. Conclusão: A utilização mensal do medicamento CERA está associada à maior qualidade de vida quando comparada a Epo-rHu. No entanto, a terapia com o novo medicamento não se mostrou mais custo-efetiva frente ao tratamento com Epo-rHu / This study sought to determine the cost-effectiveness of anemia treatment in dialysis patients for Brazilian Public Health System. Two alternatives were compared: a new drug, the Continuous Erythropoietin Receptor Activator, CERA, recently registred in Brazil, and another one, provided nowadays by the National Health System, Epo-rHu (Recombinant Human Eythropoietin). Methods: A Markov cohort of dialysis patients treated with CERA and Epo-rHu for four years was used to perform the base case analysis. The model outputs were QALYs and costs. The quality of life associated with each drug was measured by interviews applied to health care professionals. These interviews were previously submitted and approved by the local ethics committee. A sensitivity analysis was applied to the model to test it, varying the values of drugs dosage, costs, discount rate and effectiveness. Results: The average quality of life assigned by health care professionals to the patients treated with Epo-rHu, CERA and to kidney transplant receptors were respectively 6,3, 7,8 and 9,3. The model showed that Epo-rHu treatment was more cost-effective than CERA treatment. The cost-effectiveness ratio of Epo-rHu therapy was R$ 21.052,00. In addition, the cost per QALY gained of CERA therapy was R$ 72.974,00. Conclusion: Anemia treatment with CERA is associated with improvement in quality of life compared to Epo-rHu therapy. However, the new drug is not more cost-effective than the drug provided by the Brazilian Public Health System

O estudo teve como objetivo avaliar a razão de custo-efetividade, sob a perspectiva do Sistema Único de Saúde SUS, do tratamento da anemia de pacientes em Terapia Renal Substitutiva. Duas alternativas foram comparadas: um novo medicamento recentemente registrado no Brasil, o Ativador Contínuo de Receptor de Eritropoetina (Continuous Erythropoietin Receptor Activator), CERA, e outro, atualmente disponível no sistema de saúde brasileiro, a Eritropoetina Recombinante Humana - Epo-rHu. Métodos: Um modelo de Markov simulou o curso de uma coorte de pacientes em Terapia Renal Substitutiva tratados com CERA e Epo-rHu por quatro anos. A qualidade de vida associada ao uso dos medicamentos foi estimada de forma indireta, por meio de entrevista qualificada com os profissionais cuidadores, previamente submetida e aprovada pelo Comitê de Ética em Pesquisa local. Foi realizada análise de sensibilidade no modelo proposto através da variação dos parâmetros: dose dos medicamentos, custo das estratégias, taxa de desconto e efetividade utilizados para sua construção. Resultados: A média da qualidade de vida atribuída aos pacientes tratados foi 6,3 para a Epo-rHu, 7,8 para o CERA e 9,3 para os pacientes transplantados. O modelo demonstrou que a estratégia mais custo-efetiva é a terapêutica com a Epo-rHu, com um custo por QALY de R$ 21.052,00. O custo incremental por QALY ganho associado ao CERA foi de R$ 72.974,00. Conclusão: A utilização mensal do medicamento CERA está associada à maior qualidade de vida quando comparada a Epo-rHu. No entanto, a terapia com o novo medicamento não se mostrou mais custo-efetiva frente ao tratamento com Epo-rHu / This study sought to determine the cost-effectiveness of anemia treatment in dialysis patients for Brazilian Public Health System. Two alternatives were compared: a new drug, the Continuous Erythropoietin Receptor Activator, CERA, recently registred in Brazil, and another one, provided nowadays by the National Health System, Epo-rHu (Recombinant Human Eythropoietin). Methods: A Markov cohort of dialysis patients treated with CERA and Epo-rHu for four years was used to perform the base case analysis. The model outputs were QALYs and costs. The quality of life associated with each drug was measured by interviews applied to health care professionals. These interviews were previously submitted and approved by the local ethics committee. A sensitivity analysis was applied to the model to test it, varying the values of drugs dosage, costs, discount rate and effectiveness. Results: The average quality of life assigned by health care professionals to the patients treated with Epo-rHu, CERA and to kidney transplant receptors were respectively 6,3, 7,8 and 9,3. The model showed that Epo-rHu treatment was more cost-effective than CERA treatment. The cost-effectiveness ratio of Epo-rHu therapy was R$ 21.052,00. In addition, the cost per QALY gained of CERA therapy was R$ 72.974,00. Conclusion: Anemia treatment with CERA is associated with improvement in quality of life compared to Epo-rHu therapy. However, the new drug is not more cost-effective than the drug provided by the Brazilian Public Health System

L’érythropoïèse est le processus de différentiation d’un progéniteur érythroïde multipotent en globules rouges. La différentiation érythroïde est essentiellement contrôlée par le récepteur à l’érythropoïétine (EPOR). Nous avons montré que le récepteur à la transferrine de type 2 (TFR2) est un membre important du complexe formé par l’EPOR. Le TFR2 présente, comme l’EPOR une expression restreinte qui dépend du type cellulaire. Ainsi son expression n’a pu être détectée que dans le foie, l’érythron et l’intestin grêle. Le rôle du TFR2 a été exploré dans les hépatocytes et il a été montré qu’il joue le rôle d’un senseur de fer dans cette lignée et de ce fait contribue à l’homéostasie du fer. Nous avons déterminé le rôle du TFR2 dans les érythroblastes et montré que TFR2 est une protéine escorte de l’EPOR qui contribue à l’érythropoïèse in vitro et in vivo. De plus, nos travaux montrent que le TFR2 est requis pour la production de GDF15 (Growth Differentiation Factor 15) dans les érythroblastes. D’autre part nous avons démontré que la production de GDF15 est augmentée par l’EPO, la déplétion intracellulaire en fer et l’activité transactivatrice de P53. L’inhibition de l’expression de P53, réalisée au cours de l’étude de son rôle dans la production de GDF15, a révélé son implication dans l’érythropoïèse normale. Nous avons mis en évidence l’existence de plusieurs formes du TFR2. Deux d’entre elles résultent de l’utilisation de sites distincts d’initiation de la traduction. Ces deux isoformes sont régulée différemment au cours de la maturation des érythroblastes. La troisième isoforme, appelée TFR2 soluble (sTFR2), est relargée dans le plasma suite au clivage du TFR2. Nous avons montré que la production du sTFR2 est inhibée en présence du ligand de TFR2, la transferrine saturée en fer (holoTF) alors que le TFR2 est stabilisé dans ces mêmes conditions. Les rôles spécifiques des trois formes du TFR2 doivent encore être élucidés. / Erythropoiesis is the differentiation process of a multipotent erythroid progenitor into red blood cells. Erythroid differentiation is primarily controlled by the erythropoietin receptor (EPOR). We showed that the Transferrin receptor 2 (TFR2) is an important member of the EPOR complex. TFR2 has like EPOR a lineage-restricted expression and can solely be detected in the liver, erythron and small intestine. TFR2 function has been explored in hepatocytes where it plays the role of an iron sensor and contributes to iron homeostasis. We determined the role of TFR2 in erythroblasts and showed that TFR2 is an escort protein for EPOR that contributes to optimal erythropoiesis in vitro and in vivo. Moreover we evidenced that TFR2 is absolutely required for the production of Growth differentiation factor 15 (GDF15) in erythroblasts. We further demonstrated that GDF15 production is increased by EPO levels, by intracellular iron depletion as well as by P53 trans-activation activity. The inhibition of P53 expression, realized for the study of its role in GDF15 production, revealed its implication in normal erythropoiesis. We evidenced that TFR2 is expressed under several forms, two of which result from the utilization of distinct translational initiation sites. These two isoforms are differently regulated during erythroid maturation. The third form called soluble TFR2 (sTFR2) is released in the plasma after TFR2 cleavage. We showed that sTFR2 production is inhibited in the presence of TFR2 ligand, iron loaded transferrin (holoTF) whereas cell surface TFR2 expression is stabilized by holoTF. The specific roles of the three forms of TFR2 expressed by erythroblasts remain to be elucidated.