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The Israel Medical Association Journal... Nov 2002
Review
Topics: Anemia; Erythropoietin; Humans; Janus Kinase 2; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Signal Transduction
PubMed: 12489509
DOI: No ID Found -
Drug Testing and Analysis Aug 2022The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of...
The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA-stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 μl of blood, but various volumes from 20 to75 μl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 μl of serum/plasma. Different storage temperatures, from -20°C to 37°C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.
Topics: Doping in Sports; Dried Blood Spot Testing; Electrophoresis, Polyacrylamide Gel; Female; Hematinics; Humans; Male; Receptors, Erythropoietin; Substance Abuse Detection
PubMed: 35322582
DOI: 10.1002/dta.3260 -
Journal of Internal Medicine Feb 2012Complex intracellular signalling networks integrate extracellular signals and convert them into cellular responses. In cancer cells, the tightly regulated and fine-tuned... (Review)
Review
Complex intracellular signalling networks integrate extracellular signals and convert them into cellular responses. In cancer cells, the tightly regulated and fine-tuned dynamics of information processing in signalling networks is altered, leading to uncontrolled cell proliferation, survival and migration. Systems biology combines mathematical modelling with comprehensive, quantitative, time-resolved data and is most advanced in addressing dynamic properties of intracellular signalling networks. Here, we introduce different modelling approaches and their application to medical systems biology, focusing on the identifiability of parameters in ordinary differential equation models and their importance in network modelling to predict cellular decisions. Two related examples are given, which include processing of ligand-encoded information and dual feedback regulation in erythropoietin (Epo) receptor signalling. Finally, we review the current understanding of how systems biology could foster the development of new treatment strategies in the context of lung cancer and anaemia.
Topics: Anemia; Antineoplastic Agents; Cell Survival; Cytokines; Erythropoietin; Forecasting; Humans; Likelihood Functions; Lung Neoplasms; Mathematics; Models, Biological; Receptors, Erythropoietin; Recombinant Proteins; Risk Factors; Signal Transduction; Systems Biology; Transcription Factors
PubMed: 22142263
DOI: 10.1111/j.1365-2796.2011.02492.x -
Clinical Cancer Research : An Official... Sep 2009The main function of erythropoietin (EPO) is to stimulate erythropoiesis. EPO receptors (EPOR) are expressed in other cell types, including tumor cells, suggesting that...
PURPOSE
The main function of erythropoietin (EPO) is to stimulate erythropoiesis. EPO receptors (EPOR) are expressed in other cell types, including tumor cells, suggesting that the EPO/EPOR pathway governs additional cellular processes besides erythropoiesis. Recombinant EPO (rhEPO) is frequently given to anemic cancer patients, although data on clinical outcome are conflicting. In an attempt to understand these clinical data, we analyzed EPO and EPOR expression in breast cancer and evaluated EPOR as a putative prognostic and predictive marker in breast cancer patients treated with tamoxifen.
EXPERIMENTAL DESIGN
EPO mRNA/protein and EPOR mRNA were quantified by PCR and ELISA, respectively. Tissue microarrays containing 500 breast tumors from premenopausal women randomized to tamoxifen or no adjuvant treatment were evaluated for EPOR expression by immunohistochemistry. Predictive and prognostic information was evaluated using Kaplan-Meier curves and log-rank tests to estimate recurrence-free survival (RFS).
RESULTS
EPO and EPOR were expressed in cultured cells, and breast tumor specimens expressed EPOR at varying levels. Tamoxifen treatment significantly increased RFS in patients with estrogen receptor-positive/progesterone receptor-positive (ER(+)/PR(+)) tumors with low EPOR expression (P = 0.001) but had no effect on RFS in patients with tumors with high EPOR expression (P = 0.98). In the untreated cohort, RFS was significantly improved for patients with ER(+) tumors with high EPOR expression.
CONCLUSION
EPOR is abundantly expressed in breast cancer specimens. The fact that high expression of EPOR is related to an impaired tamoxifen response in ER(+)/PR(+) tumors and to improved survival in untreated patients suggests that EPOR expression in breast cancer affects tumor behavior.
Topics: Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Erythropoietin; Female; Humans; Kaplan-Meier Estimate; Prognosis; RNA, Messenger; Receptors, Erythropoietin; Tamoxifen; Tissue Array Analysis
PubMed: 19706814
DOI: 10.1158/1078-0432.CCR-08-3014 -
European Journal of Biochemistry Nov 1997Red blood cells arise continuously from pluripotent stem cells which mature and become functionally specialized upon commitment to the erythroid lineage. In mammals, the... (Review)
Review
Red blood cells arise continuously from pluripotent stem cells which mature and become functionally specialized upon commitment to the erythroid lineage. In mammals, the key regulator of this process is the hormone erythropoietin (EPO). Hormone binding to the cognate receptor, the erythropoietin receptor (EPO-R), causes receptor homodimerization and transiently triggers tyrosine phosphorylation within target cells. Although the EPO-R lacks intrinsic enzymatic activity it couples, presumably sequentially, to the protein tyrosine kinase receptor c-KIT and the cytosolic protein tyrosine kinase JAK2. Signaling through the EPO-R is promoted by tyrosine phosphorylation of the cytosolic domain and the recruitment of secondary signaling molecules such as the lipid kinase inositolphospholipid 3-kinase (phosphatidylinositol 3-kinase) and protein tyrosine phosphatase SHP-2 to the activated receptor. Complex formation of the activated EPO-R with the protein tyrosine phosphatase SHP-1 terminates signaling. In primary fetal liver cells redundant signals emanating from phosphotyrosine residues in the EPO-R support formation of erythroid colonies in vitro. However, since the last tyrosine residue in the cytosolic domain of the EPO-R, Y479, uniquely supports in the absence of other tyrosine residues an almost normal level of colony-forming unit-erythroid (CFU-E) colony formation, Y479 represents one of the key residues required in vivo for erythroid proliferation and differentiation. The signal emanating from Y479 involves sequential EPO-induced recruitment of phosphoinositol lipid 3-kinase to the EPO-R and activation of mitogen-activated-protein(MAP)kinase activity. The MAP-kinase signaling cascade could serve as an intracellular switch integrating signals mediated by several phosphotyrosine residues in the cytosolic domain of the EPO-R and provide a possible explanation for partial redundancy in signaling.
Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Phosphorylation; Phosphotyrosine; Receptors, Erythropoietin; Signal Transduction
PubMed: 9395308
DOI: 10.1111/j.1432-1033.1997.t01-1-00637.x -
CNS Neuroscience & Therapeutics Sep 2022To investigate the effect of erythropoietin (EPO) on the differentiation of neural stem cells (NSCs)/neural progenitors (NPs) in the treatment of hypoxic-ischemic injury...
AIMS
To investigate the effect of erythropoietin (EPO) on the differentiation of neural stem cells (NSCs)/neural progenitors (NPs) in the treatment of hypoxic-ischemic injury and its potential mechanisms.
METHODS
Fetal NSCs/NPs were treated with EPO after oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability, proliferation, and differentiation of NSCs/NPs were detected by CellTiter-Glo, Edu assay, flow cytometry, and quantitative real-time PCR (qPCR). Immunofluorescence staining, co-immunoprecipitation (Co-IP), and western blotting were used to test the existence of EPO receptor/β common receptor (EPOR/βCR) heterodimer on NSCs/NPs and the possible pathway.
RESULTS
EPO treatment at different time points increased cell viability without affecting proliferation. EPO treatment immediately after OGD/R promoted oligodendrocyte and astrocyte differentiation, while decreasing neuronal differentiation of NSCs/NPs. EPOR/βCR heterodimer existed on the cell surface of the fetal cortical NSCs/NPs, EPO treatment significantly increased the mRNA expression of βCR and elevated the correlation between EPOR and βCR levels. In addition, mass spectrometry analysis identified Syne-1 as a downstream signaling molecule of the EPOR/βCR heterodimer. Immunofluorescence staining and western blotting indicated that the βCR/Syne-1/H3K9me3 pathway was possibly involved in the differentiation of fetal neural stem cells into the glial cell effect of EPO.
CONCLUSION
EPO treatment immediately after OGD/R could not facilitate fetal NSCs/NPs neurogenesis but promoted the formation of the EPOR/βCR heterodimer on fetal NSCs/NPs, which mediates its function in glial differentiation.
Topics: Cell Differentiation; Erythropoietin; Neural Stem Cells; Oligodendroglia; Receptors, Erythropoietin
PubMed: 35715965
DOI: 10.1111/cns.13876 -
Blood Feb 2002Red cell development depends on the binding of erythropoietin (EPO) to receptors expressed by erythroid colony-forming units (CFUe) and the subsequent activation of...
Red cell development depends on the binding of erythropoietin (EPO) to receptors expressed by erythroid colony-forming units (CFUe) and the subsequent activation of receptor-bound Janus kinase (Jak2). Jak2 then mediates the phosphorylation of receptor tyrosine sites and the recruitment of 25 or more Src homology 2 domain-encoding proteins and associated factors. Previous studies have shown that an EPO receptor form containing Jak2-binding domains plus a single phosphotyrosine(343) (PY(343))-STAT5-binding site provides all signals needed for erythroid cell development. However, roles for PY(343) and STAT5 remain controversial, and findings regarding PY-null receptor activities and erythropoiesis in STAT5-deficient mice are disparate. To study activities of a PY-null EPO receptor in primary cells while avoiding compensatory mechanisms, a form retaining domains for Jak2 binding and activation, but lacking all cytoplasmic tyrosine sites, was expressed in transgenic mice from a GATA1 gene-derived vector as a human epidermal growth factor receptor- murine EPO receptor chimera (EE-T-Y343F). The bio-signaling capacities of this receptor form were investigated in CFUe from thiamphenicol-treated mice. Interestingly, this PY-null EPO receptor form supported CFUe development (in the absence of detectable STAT5 activation) at efficiencies within 3-fold of those levels mediated by either an EE-T-Y343 form or the endogenous EPO receptor. However, EE-T-Y343F-dependent Ter119(+) erythroblast maturation was attenuated. In tests of cosignaling with c-Kit, EE-T-Y343F nonetheless retained full capacity to synergize with c-Kit in promoting erythroid progenitor cell proliferation. Thus, EPO receptor PY-dependent events can assist late erythropoiesis but may be nonessential for EPO receptor-c-Kit synergy.
Topics: Amino Acid Substitution; Animals; Cell Division; DNA-Binding Proteins; ErbB Receptors; Erythroid Precursor Cells; Erythropoiesis; Growth Substances; Humans; Mice; Mice, Transgenic; Milk Proteins; Mutagenesis, Site-Directed; Phosphotyrosine; Proto-Oncogene Proteins c-kit; Receptors, Erythropoietin; Recombinant Fusion Proteins; STAT5 Transcription Factor; Signal Transduction; Trans-Activators
PubMed: 11806992
DOI: 10.1182/blood.v99.3.898 -
Blood Aug 2019In this issue of , Li et al show that erythropoietin receptor (Epor) expression marks the central macrophage of the erythroblastic island (EBI). Treatment with Epo...
In this issue of , Li et al show that erythropoietin receptor (Epor) expression marks the central macrophage of the erythroblastic island (EBI). Treatment with Epo increases the number of EBIs as well as the number of erythroblasts surrounding the central macrophage.
Topics: Erythroblasts; Gene Expression Profiling; Macrophages; Receptors, Erythropoietin
PubMed: 31371394
DOI: 10.1182/blood.2019001581 -
Experimental Hematology Jul 2012Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin...
Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. After stimulation with peginesatide or rHuEPO, cell lysates were prepared from UT-7/EPO cells. Liquid chromatography-tandem mass spectrometry and MesoScale arrays were used to quantify phosphorylation events. Transcriptional changes were analyzed using microarrays and quantitative reverse transcription polymerase chain reaction. Peginesatide and rHuEPO were found to regulate the tyrosine phosphorylation of an essentially equivalent set of protein substrates, and modulate the expression of a similar set of target genes. Consistent with their roles in stimulating erythropoiesis, peginesatide and rHuEPO regulate similar cellular pathways.
Topics: Cell Line; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Gene Expression Profiling; Gene Expression Regulation; Humans; Peptides; Phosphorylation; Receptors, Erythropoietin; Signal Transduction
PubMed: 22406924
DOI: 10.1016/j.exphem.2012.02.007 -
Soluble erythropoietin receptor contributes to erythropoietin resistance in end-stage renal disease.PloS One Feb 2010Erythropoietin is a growth factor commonly used to manage anemia in patients with chronic kidney disease. A significant clinical challenge is relative resistance to...
BACKGROUND
Erythropoietin is a growth factor commonly used to manage anemia in patients with chronic kidney disease. A significant clinical challenge is relative resistance to erythropoietin, which leads to use of successively higher erythropoietin doses, failure to achieve target hemoglobin levels, and increased risk of adverse outcomes. Erythropoietin acts through the erythropoietin receptor (EpoR) present in erythroblasts. Alternative mRNA splicing produces a soluble form of EpoR (sEpoR) found in human blood, however its role in anemia is not known.
METHODS AND FINDINGS
Using archived serum samples obtained from subjects with end stage kidney disease we show that sEpoR is detectable as a 27kDa protein in the serum of dialysis patients, and that higher serum sEpoR levels correlate with increased erythropoietin requirements. Soluble EpoR inhibits erythropoietin mediated signal transducer and activator of transcription 5 (Stat5) phosphorylation in cell lines expressing EpoR. Importantly, we demonstrate that serum from patients with elevated sEpoR levels blocks this phosphorylation in ex vivo studies. Finally, we show that sEpoR is increased in the supernatant of a human erythroleukaemia cell line when stimulated by inflammatory mediators such as interleukin-6 and tumor necrosis factor alpha implying a link between inflammation and erythropoietin resistance.
CONCLUSIONS
These observations suggest that sEpoR levels may contribute to erythropoietin resistance in end stage renal disease, and that sEpoR production may be mediated by pro-inflammatory cytokines.
Topics: Aged; Aged, 80 and over; Animals; Blotting, Western; Cell Line; Combined Modality Therapy; Dose-Response Relationship, Drug; Drug Resistance; Erythropoietin; Female; Humans; Interleukin-6; K562 Cells; Kidney Failure, Chronic; Logistic Models; Male; Middle Aged; Molecular Weight; Phosphorylation; Receptors, Erythropoietin; Renal Dialysis; STAT5 Transcription Factor; Tumor Necrosis Factor-alpha
PubMed: 20169072
DOI: 10.1371/journal.pone.0009246