Epo Signaling Pathway Research Articles

Angiogenic responses are enhanced by recombinant human erythropoietin in a model of periventricular white matter damage of neonatal rats through EPOR-ERK1 signaling.

Recombinant human erythropoietin (rh-EPO) has been shown to stimulate neurogenesis and angiogenesis, both of which play crucial roles in the repair of brain injuries. Previously, we observed that rh-EPO treatment effectively reduced brain damage and enhanced angiogenesis in a neonatal rat model of periventricular white matter damage (PWMD). The objective of this research is to investigate the specific mechanism through which rh-EPO regulates angiogenesis following PWMD in premature neonates. We conducted experiments utilizing a neonatal PWMD model. Following rh-EPO treatment, the levels of erythropoietin receptor (EPOR) were found to be increased in the damaged brain of rats. Although the total amount of extracellular signal-regulated kinase (ERK), a downstream protein in the EPO signaling pathway, remained unchanged, there was clear upregulation of phosphorylated ERK1 (p-ERK1) levels. The increase in levels of p-ERK1 was inhibited by an ERK kinase inhibitor, while the total amount of ERK remained unchanged. Conversely, the levels of EPOR were not affected by the inhibitor. Notably, the introduction of rh-EPO led to a significant increase in the frequency of angiogenesis-related cells and the expression levels of angiogenic factors. However, these effects were nullified when the ERK pathway was blocked. These findings indicate that rh-EPO enhances angiogenic responses through the EPOR-ERK1 pathway in a neonatal PWMD model.

Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis, allowing erythroid progenitor cells to survive and mature through their antiapoptotic action. We now know that Epo in mammals has also other functions in the brain, playing key roles in the development, maintenance, protection, and repair of the nervous system. However, the recombinant human Epo (rhEpo) has neuroprotective effects in orthoptera insects (such as grasshoppers), and this effect appears to be mediated by the cytokine receptor‐like factor 3 (CRLF3), raising interesting questions about the evolutionary origin of the Epo signaling pathway and its role in invertebrate species. Taking into account that: 1) Epo in mammals modulates the mitochondrial oxidative phosphorylation and the production of reactive oxygen species (ROS) in several tissues, including the brain; and 2) that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing and required the implementation of antioxidant systems at the cellular level; here we tested the hypothesis that activation of the “Epo‐like” system in the brain domestic crickets (Acheta domesticus) exposed to 6 % of hypoxia during 5 days, modulates mitochondrial functions for preventing against oxidative damages. To do so, we used our oxygraph‐2K system (OROBOROS) that measures the mitochondrial bioenergetics in saponin‐permeabilized tissue of 2 mg weight. Our preliminary results showed that rhEpo increased the survival of domestic crickets exposed to hypoxia by 20%. We showed also that, in normoxic animals, rhEpo increased the mitochondrial O2 consumption rate (OCR), but in hypoxic animals, rhEPO limited the increase of mitochondrial OCR. In parallel, rhEpo significantly decreased the production of ROS in hypoxia. These preliminary results suggest that rhEpo significantly improves cricket’s survival under hypoxia, by promoting a robust antioxidant effect through mitochondria. Our data also suggest that a neuroprotective “Epo‐like” endogenous molecule evolved during the “Cambrian explosion” from a urbilaterian (common to vertebrates and invertebrates) ancestor.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

Is the erythropoietin receptor the key to the identification of the central macrophage in erythroblastic islands?

Is the erythropoietin receptor the key to the identification of the central macrophage in erythroblastic islands?

Erythropoietin attenuates experimental haemorrhagic shock-induced renal damage through an iNOS- dependent mechanism in male Wistar rats

AimErythropoietin (EPO) is shown to exert protective effects on different tissues in haemorrhagic shock (HS) states. Nitric oxide (NO), as a multifunctional signaling molecule, is implicated in diverse physiologic and pathologic processes. In order to understand the exact mechanism of EPO protection, in this study we evaluated the role of different NOS enzymes in the EPO signaling pathway in male rats. MethodsRats were randomized to five groups: 1) Sham, 2) HS 3) EPO 4) L-NAME, a non-specific NOS inhibitor 5) 1400W, a specific iNOS inhibitor. HS was induced by withdrawal of 50% of total blood volume. After 2h, resuscitation was performed with the shed blood and Ringer’s lactate. In group 3, rats were treated with EPO (300IU/kg, i.v.) over 10min before HS induction. In the L-NAME and 1400W groups, L-NAME (10mg/kg, i.p.) and 1400W (2mg/kg, i.p.) were administered 30min before EPO injection. Blood and kidney tissue samples were obtained 3h after resuscitation. ResultsEPO increased the survival rate and significantly improved kidney function and histology compared to the HS group. There were less renal oxidative stress, apoptosis and systemic inflammatory responses in the EPO group. EPO increased eNOS and more abundantly iNOS mRNA expressions. L-NAME and 1400W significantly abolished all beneficial effects of EPO. ConclusionIn this in vivo animal model, we showed that EPO administration prior to HS attenuates renal injury and dysfunction in rats. The protective effects of EPO may be mediated by nitric oxide and the expression of different NOS enzymes, especially iNOS isoform.

The Role of Mitochondrial Metabolism and Redox Signaling in Iron Deficiency Anemia

Several clinical observations illustrate the link between iron and erythropoietin (EPO)-mediated signaling in committed erythroid progenitor cells. In iron deficiency anemia (IDA), erythropoiesis is blocked despite increased serum EPO concentrations. Intravenous iron improves the effectiveness of exogenous EPO in patients with EPO-refractory anemia of chronic disease. These clinical observations suggest that iron dominantly regulates EPO-receptor signaling. However, the mechanism of this iron-mediated signaling remains unclear. We recently demonstrated that 1) the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are essential in the iron- EPO-signaling pathway in erythroid progenitor cells, and that 2) isocitrate, the product of aconitase, can enhance the effectiveness of EPO during iron deficiency in vitro and in mice with IDA and in rats with the anemia of chronic inflammation. These observations suggest that isocitrate, or its derivatives that synergize with erythropoiesis stimulating agents, have important therapeutic application in the treatment of anemia. New data shows that cellular iron restriction regulates mitochondrial oxygen consumption rates (OCR) differentially over time during erythropoiesis, suggesting a novel link between mitochondrial function and erythropoeisis. It is unknown how iron deficiency induced inhibition of mitochondrial aconitase (ACO2) regulates mitochondrial metabolism during RBC production. Pilot data show that ACO2 inhibition by cellular iron deprivation or pharmacological inhibition of ACO2 decreases mitochondrial respiratory rates (RRs) and alters reactive oxygen species (ROS) production. Further, isocitrate normalizes mitochondrial RRs and ROS and restores RBC production. Importantly, disruption of mitochondrial ROS generation with a mitochondrial-specific anti-oxidant blocks RBC production and a subset of oxidant generators promote erythropoiesis. Targeted reduction of ACO2 protein expression and enzyme activity in iron replete stably transduced K562 cells decreases OCRs. This confirms the link between ACO2 and mitochondrial metabolism in human erythroid cells. These data inform our overarching hypothesis that iron-restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. The loss of this critical mitochondrial ROS signal inhibits the EPO signaling that is required for RBC production. These data also suggest that ACO2 is an iron-sensing regulator of mitochondrial metabolism and ROS signaling. 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.

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.

Regulatory Role Of Mitochondrial Aconitase and Isocitrate In Iron-Responsive, Erythropoietin Receptor Signaling During Anemia

Several clinical observations illustrate the link between iron and erythropoietin (Epo)-mediated signaling in early erythroid progenitor cells. In iron deficiency anemia (IDA) erythropoiesis is blocked at an early stage despite increased serum Epo concentrations. Intravenous iron improves the effectiveness of exogenous Epo in patients with Epo-refractory anemia of chronic disease. These clinical observations suggest that iron dominantly regulates Epo-receptor (EpoR) signaling. However, the mechanism of this iron-mediated signaling remains unclear. We recently demonstrated that 1) the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are essential in the iron- Epo-signaling pathway in early erythroid progenitors, and that 2) isocitrate, the downstream product of aconitase, can enhance the effectiveness of Epo during iron deficiency in vitro and in vivo in mice with IDA and in rats with the anemia of chronic inflammation. These observations suggest that isocitrate or derivatives of isocitrate that synergize with erythropoiesis stimulating agents have important therapeutic application in the treatment of anemia. New data from my lab also shows that cellular iron restriction regulates mitochondrial oxygen consumption rates differentially over time during red blood cell differentiation, suggesting a novel link between mitochondrial function and erythropoeisis. We also see an increase in mitochondrial superoxide anion production in iron deprived erythroid progenitors. Based on these data, we hypothesize that mitochondrial aconitase is an iron sensor that integrates mitochondrial redox signaling to EpoR signaling and subsequent red blood cell production. To test this hypothesis we are investigating the iron dependent mechanisms by which aconitase, isocitrate, reactive oxygen species and mitochondrial metabolic pathways alter EpoR signaling. 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. Disclosures:No relevant conflicts of interest to declare.

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.

Expression of erythropoietin and its receptor increases in colonic neoplastic progression: The role of hypoxia in tumorigenesis

Tissue hypoxia is a characteristic patho-physiologic property of colorectal cancer. This process may also add to a therapeutic problem of solid tumor resistance to chemo- and radiation therapy. Erythropoietin (Epo) expression is induced by tissue hypoxia. Acting via its receptor (EpoR), Epo inhibits apoptosis of erythroid cells and has been shown to rescue neurons from hypoxic damage. Increased Epo and EpoR expression has been recently described in human breast, renal and cervical carcinoma. Given the characteristic tumor diathesis present in majority of colorectal cancers, we examined whether Epo signaling may play a role in colonic neoplastic progression. Expression of Epo and EpoR was examined using immunohistochemistry in 24 cases of primary colorectal and metastatic adenocarcinomas versus adenomas and normal colonic mucosa. Immunohistochemical stains were evaluated semiquantitatively based on a four-tiered scale. Based on the combination of extent and intensity of immunoreactivity, an immunostaining score (0-300) was determined for each sample. Expression of Epo and EpoR protein and mRNA was examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, in both normal colonic tissue and carcinoma specimens in five cases. Epo expression was sequentially increased in normal colonic mucosa (8.3 ± 5.6, mean ± SEM), adenoma (26.4 ± 9.1), primary carcinoma (96.1 ± 12.8) and metastatic carcinoma (122 ± 51.3). EpoR expression was also sequentially increased in normal colonic mucosa (22.3 ± 11.8), adenoma (108.7 ± 24.2), primary carcinoma (178.7 ± 16.6) and metastatic carcinoma (220 ± 58.3) (P< 0.05 for all results). Epo and EpoR showed enhanced expression in the areas adjacent to ischemia/necrosis. Western blot and RT-PCR analysis revealed increased EpoR protein and mRNA levels in carcinoma compared to normal mucosal colon specimens. Focal stromal Epo and EpoR immunoreactivity was present in 10 and 12 cases, respectively. The uniform increase in the expression of Epo and EpoR along the colonic neoplastic sequence and further increase in ischemic/necrotic areas indicates that the Epo signaling pathway is an important component in colon carcinogenesis including possible epithelial-stromal interactions.

From Vaquez to Dameshek through JAK…2 much for polycythemia vera to be feared?

Clinical practice in modern medicine is a gift that comes from afar. It comes sometimes from a lucky discovery, or sometimes from brave attempt chasing what at that time seemed crazy theories. Anyway, the gift we currently enjoy is the result of careful observation at first, and then like a sculptor with his chisel that free from marble an hidden figure, doctors and researchers carve during the years the features of many diseases. In this number of the Internal and Emergency Medicine Journal, Landolfi and his co-workers [1] reviewed the principal characteristics of polycythemia vera (PV), starting from the history of the first published description by Luis Henry Vaquez in 1892 [2]. Since this first description something other than plethora and cyanosis were soon identified: the high risk of major thrombotic events potentially leading to death, both in arterial district (acute myocardial infarction, stroke or transient ischemic attack, TIA), and in venous district (deep vein thrombosis, pulmonary embolism, and splanchnic thrombosis). The mortality rate of PV, as reassessed in the ECLAP study [3] is about 3.7/100 patients/year due not only to the vascular complications but also for a possible evolution to malignancy. The history of PV went through the recognition of an absolute increase of blood cells, due to an abnormal erythropoiesis as the result of a pluripotential stem cell being erythropoietin hypersensitive and independent [4]. The related clinical condition must be differentiated from secondary erythrocytosis and this exercise has challenged haematologist for decades. So, like industrious sculptors, haematologist has learnt to use diagnostic criteria like chisels to carve for each patient the hidden truth of primary or secondary hyperglobulia. But not all sculptors are the same. Amedeo Modigliani, the great painter and sculptor used to say that he could see at a glance what was inside a piece of marble, and that he just had to free it. In the same way, Dameshek [5] in 1951 described his vision ‘‘from outside’’ and speculated that PV has the same origin of essential thrombocythemia (ET), chronic idiopathic myelofibrosis (IMF) and chronic myeloid leukemia (CML). What was really inside, and Dameshek had seen, was proved more than 50 years later with the discovery of the mutation at position 617 in the JH2 domain of the Janus Kinase 2 (JAK2) cytoplasmic tyrosine kinase [6–9]. This gainof-function mutation, recognizable in almost 90% of PV patients, is acquired and present in clonal hemopoietic stem cells and responsible for the activation of Epo-signaling pathway, leading to hypersensitivity and uncontrolled growth of cells. The JAK2V617F mutation is also found in about 50% of patients with ET and IMF, in atypical myeloproliferative disorders, but it has been described also in some patients with CML and in patients with portal vein thrombosis but without hematological abnormalities. From these observation, recent theories and hypothesis have been explained, such as different intensity of JAK2 signaling, presence of pre-JAK2 event that will result in different myeloproliferative disorders, different host modifier polymorphisms [10], just to cite some of them. M. Carpenedo (&) E. M. Pogliani Hematology and Bone Marrow Transplantation Unit, San Gerardo Hospital, University of Milan Bicocca, Via Pergolesi 33, 20035 Monza, Italy e-mail: mcarpenedo@katamail.com

Genome-Wide ChIP-Seq Reveals a Dramatic Shift in the EKLF Binding Profile Between Erythroid Progenitors and Erythroblasts.

Abstract 565Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of C2H2 zinc finger transcription factors. First identified as an activator of the beta-globin locus, EKLF facilitates chromatin remodeling and transcriptional activation of target genes, at least in part through recognition of a 9-base consensus motif (NCNCNCCCN). By comparing the transcriptional profiles of E13.5 wild type and Eklf-/- mice, we demonstrated that the lethal failure to complete definitive erythropoiesis in the fetal liver (FL) was due in part to dysregulation of an EKLF target gene, the cell cycle control factor, E2F2 (Pilon et al. 2008). To identify further direct targets of EKLF activation that affect erythropoiesis, we are coupling chromatin immunoprecipitation with ultra high-throughput massively parallel sequencing (ChIP-seq). ChIP-seq is increasingly being used to map protein-DNA interactions in vivo, allowing simultaneous genome-wide analysis of transcription factor occupancy, defining an ‘interactome‘. Using mice whose endogenous Eklf gene was replaced with a fully functional HA-tagged form of EKLF, chromatin was isolated at E13.5 from immature erythroid progenitors and maturing erythroblasts by ChIP. Using a highly specific high-affinity anti-HA antibody, libraries of HA-EKLF-bound chromatin were subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signature tags that were mapped to the mouse genome using the Eland software package. A control library was derived from E13.5 FL chromatin that was not enriched for HA-EKLF occupancy. For both progenitors and erythroblasts, >1.1×107tags were obtained. 72.5% and 78.7% of progenitor and erythroblast tags mapped to unique sites within the genome, respectively. The tags were highly enriched in the ∼10% of the genome within genes (genic; 42% of tags), sites ≤10 kb from the nearest gene (adjacent; 15%), as opposed to the ∼90% of the genome that is >10 kb from the nearest gene (intergenic; 22%) or in repetitive DNA (21%) p=2.2 ×10-16. Using the MACS software package clustered peaks of EKLF occupancy were identified throughout the genome, defining the EKLF ‘interactome‘. The vast majority of peaks were mapped to non-repetitive regions of the genome (98% in progenitors; 95% in erythroblasts). Progenitors contained 4,383 peaks of EKLF occupancy, while erythroblasts contained 15,396 peaks. Only 100 peaks were common between populations. This >3.5-fold increase in genomic EKLF occupancy between progenitors and erythroblasts (p=1×10-5) reflects the shift in the expression and activity of EKLF protein in erythropoiesis described previously (Bouilloux et al. 2008; Lohmann & Bieker 2008). To identify potential EKLF target genes, we partitioned the genome into 3 categories, relative to annotated RefSeq coordinates (genic) as well as adjacent and intergenic. In progenitors, the majority of EKLF binding (54%) occurred in intergenic regions, with a minority within (38%) or adjacent (7%) to genes. By contrast, the EKLF binding profile in erythroblasts was reversed, with 62% of the peaks in genic regions, and a minority at intergenic (26%) or adjacent (12%) sites.To assess the effect of this shift in EKLF binding on gene transcription, we used publicly availabel data from the inducible G1E model of erythroid maturation (GEO: GSE628) to correlate our ChIP-seq data with mRNA expression. Informatic analyses using MetaCore demonstrated that >2,200 EKLF-associated genes were differentially expressed during maturation (949 increasing expression; 1,298 decreasing expression, all p<0.05). Among progenitors, control of cell cycle S-phase entry and progression was a significantly represented network, highlighted by focal EKLF target genes like Cdk2, Cdk4, and p107, in agreement with our previous observations. Among erythroblasts, the erythropoietin (Epo) signaling pathway was most significantly represented, highlighted by focal EKLF target genes like Stat3 and Bcl-XL, reflecting the well-established importance of the Epo axis for erythroblast survival. These data indicate that shifts in EKLF occupancy during erythropoiesis correlate with distinct functional effects on gene expression. Further, these observations support a model in which transcriptional regulators (e.g., EKLF) may collect at intergenic locations when their activity is not required, but where they remain poised for rapid recruitment. Disclosures:No relevant conflicts of interest to declare.

Iron and Isocitrate Calibrate Erythropoietin Responsiveness of Erythroid Progenitors Via Aconitase Tuning of Protein Kinase C Activity.

Abstract 627Our studies have focused on signaling alterations associated with the erythroid iron deprivation response, based on the hypothesis that a discrete pathway may calibrate erythropoietin (Epo) responsiveness according to iron availability. Previously we identified aconitase enzymatic activity as a critical target in the erythroid iron restriction response and provided evidence for a non-metabolic role for its product isocitrate in coupling iron and erythropoietin signaling. The striking ability of isocitrate to reverse the erythroid iron restriction response in vitro and in vivo raised the possibility for a novel therapeutic approach to Epo-resistant anemias. New data, presented below, further support a non-metabolic mechanism of action for isocitrate rescue of erythropoiesis and identify a specific signal transduction pathway mediating cross-talk among iron, aconitase/isocitrate, and erythropoietin signaling. Two approaches addressed the significance of isocitrate metabolism by isocitrate dehydrogenase (IDH) to yield NADH, NADPH, and αa-ketoglutarate. Firstly, direct measurement of cellular NADH, NADPH, and NADP showed no significant effects due to erythroid iron deprivation or isocitrate. Secondly, we synthesized a metabolically inactive enantiomer of isocitrate, L-(+)-isocitrate, which rescued erythroid viability under iron restriction with equivalent efficiency to the metabolically active D-(−)-isocitrate. L-(+)-isocitrate, also significantly rescued erythroid differentiation under iron restriction albeit somewhat less efficiently than D-(−)-isocitrate. Interestingly, high levels of L-(+)-isocitrate occur naturally in blackberries, which have been described as a folk remedy for anemia. Further evidence for non-metabolic signaling by isocitrate arose from identification of a specific signal transduction pathway dually regulated by iron and erythropoietin. In particular, erythroid deprivation of either iron or Epo induced hyperactivation of protein kinase Cαa (PKCαa), the relevance of which was supported by multiple findings. Firstly, the hyperactivation of PKCαa during erythroid iron deprivation preceded the onset of growth and differentiation defects. Secondly, PKC inhibitors significantly reversed both viability and differentiation defects associated with iron restriction. Thirdly, isocitrate reversed PKCαa hyperactivation in cells cultured with high Epo but failed to do so in cells cultured with low Epo, correlating with the previously described inability of isocitrate to reverse the iron restriction response in low Epo. Fourthly, we and others have demonstrated direct physical interaction between mitochondrial aconitase and active forms of PKCαa/β. To assess potential relevance of iron-sulfur clusters within aconitase, IRE binding assays were conducted on progenitors −/+ iron restriction −/+ isocitrate. The patterns of IRP1 and IRP2 activation suggested that aconitase iron-sulfur clusters in the erythroid lineage are uniquely sensitive to iron deprivation and that exogenous isocitrate may act to stabilize erythroid iron-sulfur clusters in the face of iron deprivation or stress. Taken together, our results suggest a role for aconitase and isocitrate in sensing the degree of erythroid iron restriction and transducing that signal to the Epo signaling pathway. A key target appears to be PKCαa. One possible mechanism involves aconitase acting as a highly dynamic scaffold that bridges iron metabolism and Epo signaling in early erythroid development. Isocitrate may act to stabilize the active site and prevent critical conformational changes during iron restriction. The resultant conformational stability may enable tonic suppression of PKC hyperactivation and thereby permit erythropoiesis in the face of iron deprivation. Novel compounds that stabilize the activity of aconitases may offer novel therapeutic avenues for Epo-refractory anemias. Disclosures:No relevant conflicts of interest to declare.

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

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

Csk-binding Protein Mediates Sequential Enzymatic Down-regulation and Degradation of Lyn in Erythropoietin-stimulated Cells

Csk-binding Protein Mediates Sequential Enzymatic Down-regulation and Degradation of Lyn in Erythropoietin-stimulated Cells

The Critical Role of Src Homology Domain 2–Containing Tyrosine Phosphatase-1 in Recombinant Human Erythropoietin Hyporesponsive Anemia in Chronic Hemodialysis Patients

The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.

Role of the vav Proto-oncogene Product (Vav) in Erythropoietin-mediated Cell Proliferation and Phosphatidylinositol 3-Kinase Activity

The vav proto-oncogene product (Vav), which is specifically expressed in hematopoietic cells, contains multiple structural motifs commonly used by intracellular signaling molecules. Although a variety of stimuli including erythropoietin (Epo) have been shown to tyrosine phosphorylate Vav, little is known about the Vav signal transduction pathway. Here, we have investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with other proteins, using the human Epo-responsive cell line, F-36P. Immunoprecipitation and immunoblot analyses have demonstrated that Vav was associated with the Epo receptor (EpoR) in an Epo-independent manner and was tyrosine-phosphorylated after Epo stimulation. Furthermore, two phosphotyrosine proteins (pp70 and pp100) co-immunoprecipitated with the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (p85) were identified as EpoR and Vav, respectively. The interaction between Vav and p85 was shown to be mediated through the SH2 domains of p85 by an in vitro binding assay and confirmed by the presence of in vitro PI3-kinase activity associated with Vav. Treatment of the cells with antisense-vav and -p85 abrogated Epo-induced cell proliferation and PI3-kinase activity. Finally, we found that JAK2 was associated with Vav in vivo and that Vav could be tyrosine-phosphorylated by activated JAK2 in vitro. These results suggest the possible role of JAK2 for tyrosine phosphorylation of Vav and involvement of Vav and PI3-kinase in Epo-induced proliferative signals.

Role ofc-junin the Inhibition of Erythropoietin Receptor-Mediated Apoptosis

Human bone marrow cells express both a truncated and full-length form of the erythropoietin receptor (EpoR-T and EpoR-F, respectively). Transfection experiments using the murine interleukin (IL)-3-dependent cell line, Ba/F3, revealed that the cells coexpressing EpoR-F and EpoR-T (Ba/F3-FT) were more likely to undergo programmed cell death (apoptosis) than cells expressing EpoR-F (Ba/F3-FF), even in the presence of erythropoietin (Epo). When Ba/F3-FF cells were stimulated with Epo or IL-3, rapid induction ofc-myc, c-fos, c-junandjunBgenes was observed. A similar effect was also seen in IL-3-stimulated Ba/F3-Ft cells. However, in Ba/F3-FT cells expression of thec-jungene was not induced by Epo stimulation. Administration of Epo could prevent apoptosis induced by IL-3 deprivation in Ba/F3-FT cells expressing ectopic c-Jun protein. These results indicate that induction of c-Jun through the Epo signaling pathway has an important role in the inhibition of apoptosis.

Potentiation of the Erythropoietin Response by Dimethyl Sulfoxide Priming of Erythroleukemia Cells: Evidence for Interaction of Two Signaling Pathways

Erythropoietin (Epo) and dimethyl sulfoxide (DMSO) are believed to induce the differentiation of transformed erythroid cells by different signal transduction pathways. We have now obtained evidence for the interaction of these pathways. We used a Rauscher murine erythroleukemia cell line with a relatively low (8% to 10%) hemoglobinization response to Epo alone. Pretreatment of these cells for 1 day with DMSO followed by its removal and the addition of Epo resulted in a marked enhancement of the Epo specific hemoglobinization. We have designated this effect "DMSO priming." This priming effect of DMSO on the Epo response was both time-dependent and DMSO concentration-dependent. DMSO priming potentiated the Epo response in three ways. Firstly, DMSO priming increased the total number of Epo responsive cells from 8% to 10% to 40% to 60%. Secondly, DMSO priming reduced the time required to reach the optimal Epo-induced response from 4 days to 2 days. Thirdly, the Epo dose-response curve was left-shifted approximately 20-fold. DMSO priming was also associated with a marked increase in Epo receptor density characterized by an apparently new receptor population and by the appearance of positive cooperativity between receptors. Our results suggest that the DMSO priming effect is due to potentiation of the Epo signaling pathway, thus resulting in a much more rapid and dramatic Epo-induced hemoglobinization response.

Potentiation of the erythropoietin response by dimethyl sulfoxide priming of erythroleukemia cells: evidence for interaction of two signaling pathways

Erythropoietin (Epo) and dimethyl sulfoxide (DMSO) are believed to induce the differentiation of transformed erythroid cells by different signal transduction pathways. We have now obtained evidence for the interaction of these pathways. We used a Rauscher murine erythroleukemia cell line with a relatively low (8% to 10%) hemoglobinization response to Epo alone. Pretreatment of these cells for 1 day with DMSO followed by its removal and the addition of Epo resulted in a marked enhancement of the Epo specific hemoglobinization. We have designated this effect “DMSO priming.” This priming effect of DMSO on the Epo response was both time-dependent and DMSO concentration- dependent. DMSO priming potentiated the Epo response in three ways. Firstly, DMSO priming increased the total number of Epo responsive cells from 8% to 10% to 40% to 60%. Secondly, DMSO priming reduced the time required to reach the optimal Epo-induced response from 4 days to 2 days. Thirdly, the Epo dose-response curve was left-shifted approximately 20-fold. DMSO priming was also associated with a marked increase in Epo receptor density characterized by an apparently new receptor population and by the appearance of positive cooperativity between receptors. Our results suggest that the DMSO priming effect is due to potentiation of the Epo signaling pathway, thus resulting in a much more rapid and dramatic Epo-induced hemoglobinization response.