Epo Signaling Research Articles

Protein abundance of AKT and ERK pathway components governs cell type‐specific regulation of proliferation

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Erythropoietin-Mediated Neuroprotection in Insects Suggests a Prevertebrate Evolution of Erythropoietin-Like Signaling.

The cytokine erythropoietin (Epo) mediates protective and regenerative functions in mammalian nervous systems via activation of poorly characterized receptors that differ from the "classical" homodimeric Epo receptor expressed on erythroid progenitor cells. Epo genes have been identified in vertebrate species ranging from human to fish, suggesting that Epo signaling evolved earlier than the vertebrate lineage. Studies on insects (Locusta migratoria, Chorthippus biguttulus, Tribolium castaneum) revealed Epo-mediated neuroprotection and neuroregeneration. Recombinant human Epo (rhEpo) prevents apoptosis by binding to a janus kinase-associated receptor, stimulation of STAT transcription factors, and generation of factors that prevent the activation of proapoptotic caspases. Insect neurons were also protected by a neuroprotective but nonerythropoietic Epo splice variant, suggesting similarity with mammalian neuroprotective but not with homodimeric "classical" Epo receptors. Additionally, rhEpo promotes the regeneration of neurites in primary cultured insect brain neurons and after nerve crush in an in vivo preparation. In contrast to neuroprotective and regenerative effects shared with mammalian species, no evidence for a role of Epo signaling in the regulation of neuro- or gliogenesis was found in insects. Similar structural and functional characteristics of the Epo binding receptors, partly shared transduction pathways that prevent apoptosis and the functional implication in neuroprotective and neuroregenerative processes in both mammalian and insect species, suggest that Epo-like signaling was already established in their last common ancestor. Originally functioning as a tissue-protective response to unfavorable physiological situations, cell injury, and pathogen invasion, Epo was later adapted as a humoral regulator of erythropoiesis in the vertebrate lineage.

EPO Signaling Triggers Erythrocytosis By Expanding Erythrocytes and Also Subsets of Macrophages

Activated EPO receptor (EPOR) signaling (EPO-EPOR-JAK2-STATs) leads to erythrocytosis, which is defined as an absolute increase in red cell mass (RCM). Erythrocytosis is classified as primary versus secondary. Primary erythrocytosis is defined by erythroid cell-intrinsic EPO-independent mechanism due to constitutively activated EPOR signaling by gain-of function EPOR or JAK2mutations. Hypoxia inducible factor-2α (HIF-2α) is a master regulator of EPO gene expression. A defect in the oxygen-sensing VHL-PHD2-HIF-2α signaling pathway due to stress conditions or mutations leads to excess EPO production and secondary erythrocytosis (erythroid cell extrinsic EPO-dependent mechanism). It has also been suggested that a subset of macrophages play a critical role in the late stage erythropoiesis as an important erythroid cell-extrinsic factor. The role of EPOR signaling for these macrophages in the erythrocytosis context has not been fully understood.To determine the intrinsic or extrinsic regulationof erythrocytosis in vivo, we used two different mouse models of both primary and secondary erythrocytosis. We used JAK2 V617F mutant knock-in heterozygous mice (Jak2V617F mice) as a primary erythrocytosis model. To model the EPO-induced secondary erythrocytosis, we generated a liver-specific inducible human HIF-2αdouble-point-mutant (DPM) (P531A and N847A) transgenic mouse. Using liver specific Alb-Cre and Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-2α protein can be induced in a doxycycline dependent manner. We used wild-type mice for the control.Both Jak2V617F mice and doxycycline-treated Alb-Cre/LSL-rtTA-GFP/DPM (Alb-Cre/DPM) mice quickly developed a similar degree of erythrocytosis (control; Hb, 13.0 ± 1.8 g/dl; Ht, 46.2 ± 7.3%: Jak2V617F mice; Hb, 22.0 ± 0.7 g/dl; Ht, 79.7 ± 3.3%: Alb-Cre/DPM mice; Hb, 20.7 ± 2.2 g/dl; Ht, 71.4 ± 8.1%) and splenomegaly. The plasma EPO level in Jak2V617F mice was significantly decreased, while the level of EPO in Alb-Cre/DPM mice was significantly increased in comparison to the control. While the two mouse models developed similar degrees of erythrocytosis, EPO-dependent early erythroid precursors were dramatically increased in the bone marrow (BM) and spleen (SP) from Jak2V617F mice, but only modestly increased in Alb-Cre/DPM mice. This suggests that the sensitivity of the EPO-EPOR signaling pathway in erythroid cells could differ depending on whether activation is cell-intrinsic or extrinsic, and a non-erythroid population may cooperate with erythroid cells to achieve a similar degree of erythrocytosis.CD169+Vcam1+ macrophages have been shown to play a critical role in stress erythropoiesis. Thus, we analyzed the macrophage population in BM and SP. Although the frequencies of total BM macrophages did not change, CD169+Vcam1+ subpopulation, as well as total BM erythrocytes, was significantly increased in the BM macrophage population from Alb-Cre/DPM mice compared to those from the control or Jak2V617F mice. The frequencies of total SP macrophages, CD169+Vcam1+ SP macrophages, and total SP erythrocytes from both Jak2V617F mice and Alb-Cre/DPM mice were increased compared to those from the control. Notably, the increase of CD169+Vcam1+ subpopulation in the SP macrophages from Alb-Cre/DPM mice was prominent. Our results indicate that Jak2V617F could mediate cell-intrinsic activation of EPOR signaling in macrophages and induce CD169+Vcam1+ population primarily in SP but not in BM; while excessive EPO could mediate cell-extrinsic activation of EPOR signaling in macrophages and induce CD169+Vcam1+ population in both BM and SP.Taken together, using primary and secondary erythrocytosis models, we demonstrated that; 1) Jak2V617F and highly elevated EPO expression could activate EPOR signaling intrinsically or extrinsically, respectively, in both erythrocytes and macrophages, 2) both erythrocytes and macrophages cooperate to achieve an absolute increase in RCM and this could be the common underlying mechanism for erythrocytosis, 3) Extrinsic EPOR activation due to high EPO appears to induce CD169+Vcam1+ macrophages more potently than intrinsic EPOR activation in both BM and SP. Further studies are needed to understand the mechanism of how EPOR signaling induces this macrophage subpopulation, which could be a potential common therapeutic target for both primary and secondary erythrocytosis besides JAK2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Role of Pleckstrin 2 in Improved Erythropoiesis of Transferrin-Treated Beta-Thalassemic Mice

Erythropoiesis is a process during which multipotent hematopoietic stem cells proliferate, differentiate and ultimately produce enucleated reticulocytes. Terminal erythroid differentiation begins at the morphologically recognizable pro-erythroblast (pro-E) stage and is completed when orthochromatic erythroblasts (ortho-E) expel their nuclei to produce reticulocytes. Progressive differentiation between these stages occurs in homologous cell division progressively doubling proportions of pro-E, basophilic (baso-E), polychromatophilic (poly-E), and ortho-E, and multiple signaling pathways are involved in the generation of enucleated erythroid cells, including multiple steps requiring actin cytoskeleton reorganization. We have previously shown that β-thalassemic mice (th1/th1) demonstrate a disordered progression from pro-E to baso-E and that exogenous transferrin therapy restores normal proportion of early stage erythroid precursors in th1/th1 mice (Liu Blood 2013). To identify genes that play novel function in different stages of terminal erythropoiesis, we performed RNA seq analysis of sorted bone marrow pro-E from WT, th1/th1, and transferrin-treated th1/th1 mice. We identify pleckstrin-2 (plek2) as a gene of interest with a 15-fold increase in plek2 mRNA expression in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. Plek2 is an actin binding protein, like pleckstrin-1, contains a central DEP domain known to bind RacGTPase, is Epo dependent, and is expressed in all stages of terminal erythropoiesis. We evaluate plek2 mRNA and protein expression in sorted bone marrow erythroid precursors from WT, th1/th1, and transferrin-treated th1/th1 mice. Our data demonstrates a statistically significant increase in plek2 mRNA in th1/th1 relative to WT mice, with the highest expression of plek2 in poly-E, normalized in transferrin-treated th1/th1 mice (Figure 1A). A similar pattern of increased protein concentration in th1/th1 relative to WT mice and normalization in transferrin-treated th1/th1 mice is evident in sorted bone marrow samples (Figure 1B). Prior in vitro studies demonstrate that membrane localization of plek2 is required for erythroid differentiation. Thus, we performed sub-cellular fractionation in bone marrow erythroid precursors and determined for the first time that in sorted erythroblasts from WT bone marrow, plek2 is found exclusively in the cytoplasm in pro-E and in both cytoplasm and membrane from baso-E to ortho-E (Figure 2), co-localized with actin filaments in the membrane (data not shown). In contrast, sorted erythroblasts from th1/th1 bone marrow reveal membrane-associated plek2 starting from pro-E, demonstrating earlier co-localization with actin filaments (data not shown) and suggesting an earlier activation of plek2 and consequent actin cytoskeleton reorganization during erythroid differentiation in th1/th1 mice, normalized in transferrin-treated th1/th1 mice (Figure 2). Erythropoiesis involves a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, enucleation, and release into the circulation. For example, although Epo increases survival, Epo signaling also activates RacGTPases, inhibiting enucleation. Recent in vitro data demonstrates that knockdown of plek2 affected enucleation with significantly lower reticulocyte count. Although the involvement of RacGTPase in plek2-mediated erythroid differentiation has not been explored, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. Our data demonstrates that RacGTPase concentration is increased in sorted bone marrow erythroid precursors from th1/th1 relative to WT mice and normalized in transferrin-treated th1/th1 mice (Figure 1B). These results suggest that plek2 plays an important role in erythropoiesis likely as a key factor in the improved enucleation of transferrin-treated th1/th1 mice. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The clinical impact of the physiological effects of erythropoietin and erythropoietin-stimulating agents on the incidence of malignancy, and hypertension: beyond anaemia

The glycoprotein hormone erythropoietin is an essential viability and frowth factor for the erythrocytic progenitors. EPO signaling involves tyrosine phosphorylation of the homodimeric EPO receptor and subsequent activation on intracellular proteins, kinases and transcription factors. Treatment with recombinant human EPO(rHu EPO) is efficient and safe in improving the management of the anemia associated with chronic kidney disease, and allowing avoidance of transfusions with blood products. however, the unanticipated increase in mortality found in recent randomized studies is prompting a reassessment of this view. The present review will show what is known about the physiology of this plasma factor that, it is now clear, is more than just an erythrocyte production factor, and its pleitropic effects influencing the incidence of malignancy, thrombosis, hypertension and retinopathy.

Role of Iron-Responsive Mitochondrial Metabolism and ROS in Anemia

Iron and erythropoietin (Epo) are intimately linked regulators of erythropoiesis. Moderate iron restriction suppresses erythropoiesis at the Epo-dependent, CFU-E stage, without induction of apoptosis and without suppression of other hematopoietic cell lineages. Iron modulates Epo bioactivity in patients with iron deficiency anemia (IDA) and patients with anemia of chronic disease and inflammation (ACDI). To conserve iron when supplies are low, this erythroid iron restriction response reduces iron consumption by suppressing erythropoiesis. The erythroid iron sensor is unknown. Aconitases are multifunctional iron-sulfur cluster proteins localized in the cytosol (Aco1) and mitochondria (Aco2) that convert citrate into isocitrate. We have shown that iron restriction inhibits Aco2 enzymatic activity leading to suppression of erythropoiesis in vitro, and these effects are reversed by isocitrate. Isocitrate corrects IDA in mice and ACDI in rats (Bullock GC, et al. Blood. 2010;116:97-108; Richardson CL, et al. J Clin Invest. 2013 Aug 1;123(8):3614-3623). Iron restriction also alters the cross-talk between transferrin receptor and Epo receptor signaling pathways. These results suggest that Aco2 is an iron-responsive regulator of erythropoiesis. We are investigating the downstream molecular signaling mechanisms by which iron restriction induced-inactivation of Aco2 suppresses erythropoiesis. Our novel preliminary data show that mitochondrial oxidative metabolism rates change over time during erythropoiesis and that iron restriction reduces erythroid mitochondrial metabolism 4 to 7-fold compared to iron replete controls. This iron restriction induced change in respiration is associated with a significant, 1.5 to 3-fold, increase in mitochondrial superoxide production without a corresponding increase in hydrogen peroxide. Importantly, these mitochondrial alterations are reproduced by direct inhibition of aconitase with fluoroacetate (FA) and are not due to changes in mitochondrial number. Further, isocitrate reverses the effects of iron restriction or aconitase inhibition on mitochondrial metabolism and attenuates superoxide production. Based on these data and the known role of reactive oxygen species (superoxide/hydrogen peroxide) in Epo signaling, we propose the overarching hypothesis that iron restriction inhibits mitochondrial aconitase which, in turn, alters erythroid mitochondrial metabolism and ROS signaling resulting in suppression of erythropoiesis (Figure 1). We show for the first time bioenergetics profiles from iron restricted and iron replete primary human erythroid progenitor cells undergoing erythropoiesis. We also show that moderate levels of iron restriction cause mitochondrial dysfunction and alterations in mitochondrial ROS in differentiating erythroid progenitors. The clinical relevance of this project lies in its potential for the development of new iron-free agonists and antagonists of red blood cell production. Agonists may benefit patients with anemia due to iron deficiency or chronic inflammation and antagonists may benefit patients with myeloproliferative neoplasms.Figure 1:Proposed mechanism of iron-dependent regulation of erythropoiesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Cell Kinase (HCK) Is Involved in Myelodysplastic Syndromes and Acute Leukemia Pathophysiology By Modulating PI3K Pathway and Enhancing Erythroid Differentiation

Introduction: Src family non-receptor tyrosine kinase regulates diverse cellular responses mainly by activating PI3K signaling, an important pathway that contributes to many cancers pathology. HCK, a Src family member, is restricted expressed in hematopoietic cells; however, high levels of HCK have been reported in solid tumors and hematologic neoplasms and have been associated with a worse prognosis. Identification of unbalanced pathways and upregulated proteins and a better knowledge of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) pathogenesis could help identify potential druggable targets and new prognostic biomarkers. The clinical and biological role of HCK in MDS and AML pathophysiology has not yet been elucidated and may be a novel therapeutic approach.Aims: To evaluate HCK expression in hematopoietic stem cells (HSC/CD34+) and in total bone marrow (BM) cells from healthy donors and MDS and AML patients. We further attempted to analyze the HCK knockdown and drug inhibition on differentiation, proliferation, apoptosis and migration in HSC/CD34+ and in a panel of human leukemia cell lines (KG1a, U937, HL60, P39).Materials and Methods: A total of 34 healthy donors and 139 patients at diagnosis (MDS=75 [low-risk=46, high-risk=29], AML with Myelodysplastic related changes (AML-MRC)=21 and de novo AML=43), were included in the study. HCK gene and protein expression was analyzed by qPCR and Western blot. Results are expressed as median (minimum–maximum) after appropriate statistical analysis. For functional analysis, HCK was inhibited with specific lentiviral vector system in HSC/CD34+ (isolated from umbilical cord blood units and BM), KG1a, U937, HL60 and P39 cells. Apoptosis was evaluated by Annexin-V/PI, cell growth by CellTiter assay, migration by Transwell, CXCR4 expression and actin polymerization by flow cytometry. We also verified the effects of a HCK specific inhibitor, ASN05260065, kindly provided by ASINEX and Dr Maurizzio Bottas.Results: HCK mRNA were significantly increased in HSC/CD34+ from patients with MDS (4.87 [0.03-14.75]; P=.003) and AML (8.36 [0.70-24.47]; P=.003) compared to healthy donors (1.19 [0.06-3.02]). When patients were stratified according to WHO classification, HCK expression was significantly higher in low-risk MDS compared to high-risk and AML-MRC (8.10 [2.30–14.75] vs 3.22 [0.03–6.33] and 1.02 [0.70–2.78], P<.001). Similar results were found in BM cells: HCK transcripts were significantly increased in MDS compared to healthy donors, and in low-risk compared to high-risk MDS (P<.0001). Interestingly, 5 of 5 MDS patients showed a significant decrease in HCK expression after disease progression. Also, a negative correlation was observed between BM blast percentage and HCK expression in MDS patients. In HSC/CD34+ cells, BFU-E colony number was significantly lower after HCK inhibition. HCK depletion reduced cell death in HSC/CD34+, P39 and KG1a cells and diminished proliferation in KG1a and U937 cells. Moreover, HCK knockdown decreased the phosphorylation of AKT, mTOR (PI3K pathway members) and ERK and treatment of HCK silenced cells with PI3K inhibitors had no synergic effect. Confirming the HCK lentivirus silencing results, HCK specific inhibitor treatment downregulated PI3K and ERK signaling. In addition, HCK inhibition significantly reduced migration towards CXCL12 chemoattractive stimuli by regulating CXCR4 expression and F-actin polymerization. Attempts have been made to transduct CD34+ from low-risk MDS patients however more than 80% cells were apoptotic and impossible to perform further assays.Conclusions:HCK is overexpressed in HSC/CD34+ and BM cells from MDS, mainly in low-risk MDS, and de novo AML. During MDS progression, HCK expression reduction correlates with increasing blast percentage and could indicate less dependence on microenvironment. HCK inhibition reduced BFU-E colony formation and our previous results showed that HCK expression increases during erythroid differentiation of non-transduced low-risk MDS CD34+ cells. Thus, HCK seems to play a role as a downstream effector in erythropoietin (Epo) signaling which could explain the higher HCK expression in low-risk MDS. Besides, HCK is an upstream regulator of PI3K and MAPK pathway that enhances cell apoptosis, survival and proliferation. Our study described new insights to the role of HCK in Epo/PI3K signaling and in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Epo-induced erythroid maturation is dependent on Plcγ1 signaling.

Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Binding of erythropoietin (Epo) to its receptor (EpoR) is required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Janus kinase 2 (Jak2) tyrosine kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR-Jak2 independently of Stat5. Plcγ1-deficient pro-erythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined HSCs (Lin(-)Sca1(+)KIT(+)CD48(-)CD150(+)) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation, we assessed changes occurring at the global transcriptional and DNA methylation level after inactivation of Plcγ1. The top common downstream effector was H2afy2, which encodes for the histone variant macroH2A2 (mH2A2). Inactivation of mH2A2 expression recapitulated the effects of Plcγ1 depletion on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target mH2A2, as a 'non-canonical' Epo signaling pathway essential for erythroid differentiation.

Poster Presentations

Administration of recombinant human erythropoietin (rHuEpo) improves performance and hence is subject to abuse by athletes. The detection of rHuEpo doping remains a major challenge at present. The aim of the current study was to investigate whether circulating microRNA (miRNA) can be used for detecting r-HuEpo doping. Twenty trained males received rHuEpo injections of 50 IU∙kg -1 body mass every two days for 4 weeks. Blood was obtained 2 weeks before, during and 4 weeks after administration. For this pilot analysis, plasma miRNA expression was assessed at selected time points using the Affymetrix GeneChip 3.0 and the miScript 384 HC PCR Array (Qiagen). For the Affymetrix microarray data, GC content adjustment was carried out prior to background correction and quantile normalization. For the Qiagen PCR Array data, raw CT values exported from the RQ manager were firstly normalized to the spike-in control (i.e. cel-miR-39-3p), then to the mean of commonly expressed miRNAs with a CT < 35 prior to formal analysis. 1733 human miRNAs are present on the GeneChip 3.0. Eighty-two of the 1733 miRNAs were commonly expressed across all samples and 773 miRNAs were found in at least one sample. Six miRNAs showed significant fold changes following r-HuEpo, with 5 miRNAs up-regulated (fold change ranging from 2.03-3.07, P < 0.05) and 1 miRNA down-regulated (fold change: 2.56, P = 0.042). Out of 372 human miRNAs present (Qiagen), 205 human miRNAs were detected in all samples and 164 miRNAs were found in at least one sample. Two miRNAs were differentially expressed (fold changes: 3.4 - 3.84, P ≤ 0.03) following r-HuEpo. For significantly expressed miRNAs identified by both methods, 6 common miRNAs were found to be either predicted or validated miRNAs potentially involved in Epo signalling pathways. Analysis of identified miRNAs in the remaining samples will be required in order to determine whether circulating miRNAs can be effectively used for r-HuEpo doping detection. miRNAs isolated from red blood cells before and after r-HuEpo are also currently being analysed using next generation sequencing.

Treatment with erythropoiesis-stimulating agents in chronic kidney disease patients with cancer

Treatment of anemia remains an important component in the care of patients with nondialysis chronic kidney disease (CKD) and end-stage renal disease (ESRD). Erythropoietin-stimulating agents (ESAs) remains a key anemia treatment strategy in this patient population. However, anemia management in this group can become more complicated by prior or current history of malignancy. There has been a great deal of work both scientifically and in clinical trials in oncology that have revealed certain concerns and risks of ESA use in patients with cancer. In this review, we will bring together knowledge from nephrology and oncology literature to help nephrologists understand the implications for ESA treatment when CKD/ESRD is complicated by cancer. We also suggest an approach to the management of anemia in this patient group with active or previous malignancy.

Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis

AbstractErythropoietin (Epo) binding to the Epo receptor (EpoR) elicits downstream signaling that is essential for red blood cell production. One important negative regulatory mechanism to terminate Epo signaling is Epo-induced EpoR endocytosis and degradation. Defects in this mechanism play a key role in the overproduction of erythrocytes in primary familial and congenital polycythemia (PFCP). Here we have identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85 regulatory subunit of PI3K, which binds to phosphotyrosines on EpoR. Ubiquitination allows p85 to interact with the endocytic protein epsin-1, thereby driving EpoR endocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant devoid of ubiquitin-interacting motifs all compromise Epo-induced EpoR internalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, or internalize on Epo stimulation and exhibit Epo hypersensitivity. Similarly, knockdown of Cbl also causes Epo hypersensitivity in primary erythroid progenitors. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization and EpoR internalization and normalizes Epo hypersensitivity. Our results uncover a novel Cbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP.

Conflicts Of Interest In Pharmaceutical Sponsored Research On Erythropoietin Receptors In Cancer: An Updated Analysis

BackgroundFinancial conflicts of interests in clinical studies have been a perennial subject of investigation because of their potential to harm the public's health. In 2010, we reported the first systematic analysis examining such conflicts in preclinical research, identifying that academic researchers without financial conflicts of interest significantly differed from academic researchers with financial conflicts of interest as well as scientists employed by manufacturers of erythropoiesis stimulating agents (ESAs). Those with no conflict of interest identified functional erythropoietin (Epo) receptors on tumor cells and that these receptors when activated carried out detrimental cell signaling and tumor promotion effects. The analysis was based on published studies from 1993 to 2008. In 2008, an NCI-sponsored conference with 14 academic scientists and 6 manufacturer employed-scientists restated these findings. We now review the status of the peer-reviewed published literature that has been disseminated following the NCI conference. MethodsArticles identified in MEDLINE and EMBASE databases (2008-2012) investigating preclinical findings were reviewed for information on Epo receptors, signaling events, cellular function, and study conclusions. Study findings were classified into 3 groups according to funding source. (1) Academic-based studies authored by investigators without manufacturer funding [13 studies], (2) academic-based studies authored by investigators with funding from ESA manufacturers [8 studies], and (3) industry-based studies authored by more than 75% of investigators employed by ESA manufacturers [2 studies]. ResultsStudies authored by investigators in groups 1 and 2 report: Epo-induced signaling events in tumor cells (84.6% and 100.0%, respectively) and conclude that ESAs could be clinically harmful (83.3% and 57.2%, respectively); while no investigators in group 3 report Epo-induced signaling events in tumor cells or conclude that ESAs could be clinically harmful. Using univariate optimal discriminate analysis, 6/9 studies authored by investigators in groups 2 and 3 found no harmful changes in cellular function and is statistically different from investigators in group 1 who found harmful changes in cellular function in 11/12 studies (p<.02). ConclusionsConvergence has occurred over time regarding the harmful conclusions of ESAs and cancer progression among academic scientists with versus without financial conflicts of interests. However scientists employed by ESA manufacturers still report no detrimental basic science findings of ESAs on tumor cells. This claim is of considerable importance to the ESA manufacturers, as FDA advisories have indicated that should detrimental preclinical effects of ESAs be found on certain tumor cell lines, then the advisory committees may consider particular caution in the use of ESAs among persons with these specific tumor types. Disclosures:Bennett:Amgen: Consultancy, Research Funding.

Erythropoietin Potentiates The Immune System Yet Induces Bone Resorption In a Myeloma Mouse Model

BackgroundMultiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells and monoclonal gammopathy. It is associated with various clinical manifestations including bone disease and anemia. Clinical introduction of recombinant human erythropoietin (rHuEPO) and its derivatives has been a breakthrough in treating patients with anemia, mainly those with chronic kidney disease and cancer patients on chemotherapy. EPO was found to have additional biological effects, including potentiation of both humoral and cell-mediated immune responses. Our studies and those of others, have shown that EPO treatment for anemia in MM patients was associated with general improved immunological functions and immune-mediated modulation (suppression) of MM. A role for EPO in bone metabolism was previously suggested, but its effects were often controversial and the underlying mechanisms remain to be resolved. ObjectivesTo determine the immunological and skeletal effects as well as the underlying mechanisms of EPO signaling in the 5T33 MM mouse model. ResultsEPO administration led to a 50% decrease in the levels of the MM pathological κ light chain while attenuating the decrease in normal IgA. These immunoglobulin changes were accompanied by a 70% elevation (p=0.015) of B220+IGM+IgD- immature B cells in the BM of EPO-treated MM mice, compared to diluent-injected MM mice (9.8%±0.91 versus 5.68%±1.19 of B220-gated cells, respectively). We also found a 45% increase (p=0.01) in the level of BM macrophages (F4/80+CD11b+) following EPO administration in both healthy and MM mice. Moreover, stimulation in vitro of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of the 5T33 MM cells.Th17 cells play an important role in MM pathobiology. Our data demonstrate that in MM mice, rHuEPO administration induces a 70% decrease (p=0.021) in the expression level of RORγt, a Th17 hallmark transcription factor. These findings were associated with a 4-fold increase (p<0.001) in the transcript levels of the Th1-associated cytokine IFN-γ, in the BM of EPO-treated MM mice versus their diluent-injected MM counterparts. As MM is typically associated with a Th2 response and since Th17-associated cytokines in MM patients are suggested to inhibit the Th1 response, EPO may thus have a beneficial role in MM, by shifting the balance towards a Th1 response. Collectively, these findings demonstrate that EPO treatment potentiates humoral immune responses, and limits MM progression.Bone disease is a major component of MM and is associated with significant morbidity. High-resolution CT analysis of the distal femurs confirmed bone loss in 5T33 MM mice. The femur bone density of 5T33 MM mice was 30% lower (p=0.04) than that of their healthy counterparts (3.1%±0.003 versus 4.4%±0.008, respectively). Unexpectedly, EPO treatment of MM mice, in comparison to diluent-injected MM mice, induced a 35% reduction (p=0.004) in trabecular bone density (3.1%±0.003 and 2%±0.007, respectively) and 41% (p<0.001) reduction in trabecular number (2.62mm-1±0.23 and 1.54mm-1±0.266, respectively). This was only partly compensated by a 19% increase (p=0.006) in trabecular thickness (0.043mm ±0.005 versus 0.036mm ±0.003 in controls) in EPO-treated MM mice. Bone loss correlated with a 50% increase in transcript levels of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in BM of EPO-treated MM mice compared to diluent-injected MM mice. In line with the premise that EPO-induced bone loss is mediated by increased osteoclastogenesis, we found an EPO-associated elevation in BM osteoclast progenitors, as determined by RANK and cFms immunostaining (cFms+/RANK−, 1.7 fold, p=0.01; cFms+/RANK+, 2.7 fold, p=0.007). ConclusionsWe show that in MM, EPO acts as a double-edged sword, by stimulating the immune response against MM on one hand, but also accelerating bone resorption on the other, which further alters the already compromised bone tissue. A better understanding of the osteoimmunological roles of EPO may advocate for the use of rHuEPO along with targeted bone protective treatment in MM patients, to attenuate the anemia and MM progression, while also preventing bone damage.ND-U and SH-B have contributed equally. Supported by the Multiple Myeloma Research Foundation (DN). Disclosures:No relevant conflicts of interest to declare.

Stimulating erythropoiesis in neonates

Stimulating erythropoiesis in neonates

Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling

AbstractLyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lynup/up) on erythropoiesis and Epo receptor signaling. Adult Lynup/up mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn+/up mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn+/up erythroid cell lines and primary CD71+Lynup/up erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated-binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn+/up cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects.

Human erythropoietin increases the pro-angiogenic potential of A2780 ovarian adenocarcinoma cells under hypoxic conditions

Erythropoietin (Epo) is a key regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of the recombinant protein, it is widely used to treat various types of anemias, including that associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Our previous studies confirmed the presence of Epo receptors (EpoRs) in ovarian adenocarcinoma cell lines and demonstrated that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting both enhanced Epo signaling and increased paclitaxel resistance. In the present study, we carried out a series of experiments to analyze the pro-angiogenic potential of Epo-treated A2780 and SKOV-3 cells. Our studies revealed that conditioned media of Epo-treated A2780 cells had a stimulative effect on human umbilical vein endothelial cells (HUVECs). This effect was only seen when A2780 cells were incubated under hypoxic conditions. Furthermore, Epo increased the secretion of interleukin (IL)-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, GM-CSF and interferon-γ by A2780 cells that grew in hypoxic conditions. In this regard, conditioned media of hypoxic and Epo-treated A2780 cells induced a significant phosphorylation of STAT-5 in HUVECs. Our results may have important implications for ovarian cancer patients receiving Epo.

Layered Signaling Regulatory Networks Analysis of Gene Expression Involved in Malignant Tumorigenesis of Non-Resolving Ulcerative Colitis via Integration of Cross-Study Microarray Profiles

BackgroundUlcerative colitis (UC) was the most frequently diagnosed inflammatory bowel disease (IBD) and closely linked to colorectal carcinogenesis. By far, the underlying mechanisms associated with the disease are still unclear. With the increasing accumulation of microarray gene expression profiles, it is profitable to gain a systematic perspective based on gene regulatory networks to better elucidate the roles of genes associated with disorders. However, a major challenge for microarray data analysis is the integration of multiple-studies generated by different groups.Methodology/Principal FindingsIn this study, firstly, we modeled a signaling regulatory network associated with colorectal cancer (CRC) initiation via integration of cross-study microarray expression data sets using Empirical Bayes (EB) algorithm. Secondly, a manually curated human cancer signaling map was established via comprehensive retrieval of the publicly available repositories. Finally, the co-differently-expressed genes were manually curated to portray the layered signaling regulatory networks.ResultsOverall, the remodeled signaling regulatory networks were separated into four major layers including extracellular, membrane, cytoplasm and nucleus, which led to the identification of five core biological processes and four signaling pathways associated with colorectal carcinogenesis. As a result, our biological interpretation highlighted the importance of EGF/EGFR signaling pathway, EPO signaling pathway, T cell signal transduction and members of the BCR signaling pathway, which were responsible for the malignant transition of CRC from the benign UC to the aggressive one.ConclusionsThe present study illustrated a standardized normalization approach for cross-study microarray expression data sets. Our model for signaling networks construction was based on the experimentally-supported interaction and microarray co-expression modeling. Pathway-based signaling regulatory networks analysis sketched a directive insight into colorectal carcinogenesis, which was of significant importance to monitor disease progression and improve therapeutic interventions.

EPO signaling and hematopoietic expansion as causes of osteoporosis in a thalassemia mouse model

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

How a Blood Cell Intracellular Signal Keeps Blood Flowing

‘‘Epo,’’ or erythropoietin, is not just a performance-enhancing drug for endurance athletes. It is an essential hormone that regulates the rate of erythropoiesis, or creation of new red blood cells. Basal Epo signaling ensures that such cells are made constantly at a low level, to replace wornout cells. Certain stresses, such as hypoxia, increase Epo and lead to a steep increase in erythropoiesis, a phenomenon known to mountain climbers, who adapt to the low oxygen at high altitudes by packing more red blood cells into their blood. The Epo signaling cascade begins when the hormone binds to the Epo receptor. This ultimately leads to phosphorylation of a transcription factor called Stat5, which turns on a suite of genes controlling cell proliferation. A key question in understanding the Epo-Stat5 system is whether and how basal and stress-induced signaling differ. In a new study in this issue of PLOS Biology, Emelinda Porpiglia, Merav Socolovsky, and colleagues set out to answer that question. The authors used fluorescently labeled antibodies to tag phosphorylated Stat5 in erythropoeitic liver cells of mouse embryos. They also used a variety of cell surface markers to distinguish early versus late erythroblasts (cells that differentiate into red blood cells). They characterized three aspects of Stat5 signaling within Epostimulated cells: the median fluorescence from all cells within a subpopulation of erythroblasts, the fraction of those cells that were engaged in Stat5 signaling, and the level of phosphorylated Stat5 within signaling cells. They found that Stat5 signaling was highest in precursor cells, and declined with maturation. Furthermore, precursors exhibited an ‘‘analog,’’ graded increase in signaling in response to rising Epo levels. This graded increase was a combination of an increase in the number of signaling cells, and an increase in the signaling intensity within each cell. In contrast, older cells, which overall had much lower Stat5 signaling, responded to an increase in Epo by increasing the number of signaling cells, but within each cell, the signal intensity was binary, either ‘‘on’’ or ‘‘off,’’ with little change in intensity in the ‘‘on’’ position. What regulates this transition from analog to binary response? In large part, they found, the ability to mount a highintensity and graded response was due to larger amounts of Stat5 protein in earlier cells, rather than differences in Epo receptor level. When the authors added exogenous Stat5 to older cells, they too were able to signal at the higher level and in a graded way. Conversely, in cells with a mutant Epo receptor unable to bind Stat5, only basal-level, binary signaling was present, similar to the older cells. A binary signal, the authors argue, provides an ideal way to filter out the noise at low incoming Epo signal strength, while the analog response at higher Epo concentrations provides the cell with a more dynamic ability to respond to varying levels of stress. Additionally, they found, part of the stress response was an increased production of transferrin receptor (CD71), probably to help bring in extra iron to make more hemoglobin. Understanding this system is important not only for a better theoretical understanding of the dynamics of cell signaling. Targeting the high-level Stat5 signal, while maintaining the low-level one, may be a viable strategy for treating myeloproliferative disorders, including some types of leukemia, in which Stat5 is constitutively active.

Erythropoietin in Brain Development and Beyond

Erythropoietin is known as the requisite cytokine for red blood cell production. Its receptor, expressed at a high level on erythroid progenitor/precursor cells, is also found on endothelial, neural, and other cell types. Erythropoietin and erythropoietin receptor expression in the developing and adult brain suggest their possible involvement in neurodevelopment and neuroprotection. During ischemic stress, erythropoietin, which is hypoxia inducible, can contribute to brain homeostasis by increasing red blood cell production to increase the blood oxygen carrying capacity, stimulate nitric oxide production to modulate blood flow and contribute to the neurovascular response, or act directly on neural cells to provide neuroprotection as demonstrated in culture and animal models. Clinical studies of erythropoietin treatment in stroke and other diseases provide insight on safety and potential adverse effects and underscore the potential pleiotropic activity of erythropoietin. Herein, we summarize the roles of EPO and its receptor in the developing and adult brain during health and disease, providing first a brief overview of the well-established EPO biology and signaling, its hypoxic regulation, and role in erythropoiesis.