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At least 19 recordsLinked to original sources
PubMed1992

The International Standard for Recombinant DNA-derived Erythropoietin: collaborative study of four recombinant DNA-derived erythropoietins and two highly purified human urinary erythropoietins.

The International Standard (IS) for Recombinant DNA-Derived (rDNA) Erythropoietin (EPO) (in ampoules coded 87/684) and three other rDNA EPO preparations in ampoules coded 87/690, 87/696 and 88/574 respectively, were compared with two preparations of highly purified human urinary (HU) EPO and the 2nd International Reference Preparation of Human Urinary Erythropoietin for Bioassay (2nd IRP) by 26 laboratories in 11 countries using a wide range of in-vivo and in-vitro bioassays and immunoassays. These EPO preparations were also compared by electrophoresis and isoelectric focusing. Estimates of EPO content in terms of the 2nd IRP by all in-vivo bioassay methods gave combined unweighted geometric means (with 95% fiducial limits) of: 86 (75-99) IU/ampoule for the IS, 81 (70-94) IU/ampoule for 87/690, 58 (48-71) IU/ampoule for 87/696 and 120 (100-143) IU/ampoule for 88/574. Mean estimates of EPO content in terms of the 2nd IRP by in-vitro bioassays (except receptor assays) were larger than, and those by immunoassays were similar to, the mean estimates by in-vivo bioassays. The use of purified rDNA or HU EPO as standards in place of the 2nd IRP reduced the inter-laboratory variability of estimates of purified EPO preparations by in-vivo and in-vitro bioassays and by immunoassays, and reduced the variability of overall mean estimates for each of these preparations between the three types of method. The inter-laboratory variability of immunoassay estimates of human serum EPO was similar whether the 2nd IRP or one of the purified EPOs was used as standard. Significant differences in in-vivo and in-vitro biological, immunological and physicochemical properties were found between these four rDNA EPO preparations and between them and the HU EPO in the two purified preparations and in the 2nd IRP. There were also differences between the immunoreactivities of the two serum EPO samples included in the study, and between them and the immunoreactivities of the purified EPOs. The differences between rDNA EPOs appeared to be related to differences between the cells used for their biosynthesis, but may also be the result of differences in purification methods and of inter-batch variations. Significant differences in assay specificity were observed within each of the three general types of method. The specificity of the in-vivo bioassays was influenced by the route of hormone administration. The specificities of the mouse spleen cell in-vitro bioassays differed from that of the mouse spleen receptor-binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Assay↗
PubMed2001

A novel mechanism of cooperation between c-Kit and erythropoietin receptor. Stem cell factor induces the expression of Stat5 and erythropoietin receptor, resulting in efficient proliferation and survival by erythropoietin.

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

Animals↗
PubMed2005

Erythropoietin and erythropoietin receptor expression after experimental spinal cord injury encourages therapy by exogenous erythropoietin.

OBJECTIVE: Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions. Because the expression of the EPO and EPO-R network is poorly investigated in the central nervous system, the aim of the present study was to investigate whether the resident EPO and EPO-R network is activated in the injured nervous system. METHODS: A well-standardized model of compressive spinal cord injury in rats was used. EPO and EPO-R expression was determined by immunohistochemical analysis at 8 hours and at 2, 8, and 14 days in the spinal cord of injured and noninjured rats. RESULTS: In noninjured spinal cord, weak immunohistochemical expression of EPO and EPO-R was observed in neuronal and glial cells as well as in endothelial and ependymal cells. In injured rats, a marked increase of expression of EPO and EPO-R was observed in neurons, vascular endothelium, and glial cells at 8 hours after injury, peaking at 8 days, after which it gradually decreased. Two weeks after injury, EPO immunoreactivity was scarcely detected in neurons, whereas glial cells and vascular endothelium expressed strong EPO-R immunoreactivity. CONCLUSION: These observations suggest that the local EPO and EPO-R system is markedly engaged in the early stages after nervous tissue injury. The reduction in EPO immunoexpression and the increase in EPO-R staining strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration.

Aneurysm↗
PubMed2003

Erythropoietin and the erythropoietin receptor are expressed by papillary thyroid carcinoma from children and adolescents. Expression of erythropoietin receptor might be a favorable prognostic indicator.

Erythropoietin (EPO) and the EPO receptor (EPO-R) have been implicated in solid tumors of the brain, breast, kidney and female genital tract. Based on their expression by a variety of tumors, we hypothesized that EPO and/or EPO-R might be expressed by thyroid cancers. To test this, we determined EPO and EPO-R expression by immunohistochemistry in 17 papillary thyroid carcinomas (PTC) from children and adolescents. Only a minority of PTC (4/17, 24%) expressed EPO, and there were no significant differences between the PTC that did or did not express EPO. In contrast, EPO-R was detected in the majority of PTC (11/17, 65%). The average tumor size (1.5 +/- 0.8 cm), MACIS score (3.6 +/- 0.2) and risk of recurrence (0/11) for the EPO-R(+) PTC were significantly less than those for PTC that failed to express EPO-R (average tumor size = 3.6 +/- 2.4 cm, p = 0.021; average MACIS score = 4.3 +/- 0.7, p = 0.004; recurrence = 3/6, p = 0.029). We conclude that the majority of PTC from children and adolescents express EPO-R, a finding associated with favorable prognostic indicators and a lower risk of recurrence.

Adolescent↗
PubMed1996

Comparison of dose requirement, serum erythropoietin and blood pressure following intravenous and subcutaneous erythropoietin treatment of dialysis patients. IV and SC erythropoietin.

OBJECTIVE: The purpose of the study was to investigate the effect of route of administration of erythropoietin (EPO) on the dose requirement in dialysis patients after intravenous (IV) and subcutaneous (SC) therapy. METHODS: The study was performed as a single centre, prospective, open, combined parallel and cross-over study of 50 dialysis patients, consecutively randomised to IV or SC treatment with EPO. The initial dose was 49 U.kg-1 3-times weekly, adjusted to increase haemoglobin (Hgb) from a median 5.3 mmol.1(-1) to a target of haemoglobin 6.5-7.5 mmol.1(-1). After reaching the target level, the haemoglobin was maintained for 4 months (Period 1). Then IV and SC treatment was switched for a further 4 months (Period 2). The study included high risk patients. The adjustment period was completed by 38 patients, Period one by 32 patients (IV/SC = 15/17; male/female = 19/13; age = 54 (24- 71) y), and Period two by 22 patients. RESULTS: No significant difference was found between the two groups in the reticulocyte response, the rate of Hgb increase (IV 0.7 versus SC 0.5, mmol.1(-1). month-1), time to reach target level (IV 43 versus SC 60 days), or total EPO dose per increase in haemoglobin to target level (IV 663 versus SC 946 (U.kg-1) per (mmol Hgb.1(-1)). The overall median maintenance dose during the last month of the two four month periods was 105 (range IV 51-336) U.kg-1.w-1 and SC 104 (range 21-321) U.kg-1.w-1. Through serum EPO levels were significantly higher during SC treatment. The blood pressure did not change significantly from the base level after either route of administration; start 133/80 versus 143/80 mmHg, target 127/78 versus 154/85 mmHg, and maintenance period 140/84 versus 142/85 mmHg. Thus, three-times weekly IV or SC EPO did not differ significantly in efficacy or in the effect on blood pressure in dialysis patients.

Adult↗
PubMed1991

Erythropoietin in chronic obstructive pulmonary disease. Relationship between serum erythropoietin, blood hemoglobin and lung function--effect of the calcium antagonist isradipine on serum erythropoietin.

The relations between serum-erythropoietin (se-EPO), blood hemoglobin and lung function were investigated in patients with chronic obstructive pulmonary disease (COPD). In a randomized, double-blind, placebo-controlled design, the hypothesis was tested that se-EPO might increase after treatment with a calcium antagonist. In 18 patients with COPD, median se-EPO (19.2 mU ml-1) was in the low normal range. The correlations between se-EPO and blood hemoglobin (p = -0.63, p = 0.024) and between se-EPO and lung function indices were negative (n.s.) and the correlation between blood hemoglobin and lung function indices (n.s.) were positive. The hypothesis is proposed that the normal hemoglobin found in most patients with COPD is a result of a balance between a trend towards a decreased red cell mass, as found in chronic diseases, and a trend towards an increased red cell mass due to the erythropoietic effect of EPO. Se-EPO was not changed by the calcium antagonist, isradipine.

Aged↗
PubMed1989

Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration.

In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP-producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP-producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP-producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.

Acute Disease↗
PubMed2005

Relation of erythropoietin and erythropoietin receptor expression to hypoxia and anemia in head and neck squamous cell carcinoma.

PURPOSE: The use of erythropoietin in head and neck squamous cell carcinoma (HNSCC) has been associated with poor survival. This study examines the protein and mRNA expression of erythropoietin and erythropoietin receptor in HNSCC and their relation to hypoxia, hemoglobin (Hb), and clinical outcome. EXPERIMENTAL DESIGN: The immunohistochemical expression of erythropoietin and erythropoietin receptor was assessed in 151 cases of HNSCC. Expression was compared with the hypoxia-dependent proteins hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase-9 (CA-9) and correlated with clinical outcome. The mRNA expression of erythropoietin and erythropoietin receptor was measured in paired samples of HNSCC. RESULTS: Erythropoietin and erythropoietin receptor were expressed in 95% and 99% of tumors, respectively. Using a weighed expression score, there was a positive correlation between erythropoietin and erythropoietin receptor expression (r = 0.18, P = 0.03). HIF-1alpha (r = 0.38, P < 0.01) and CA-9 (r = 0.26, P = 0.002) correlated with erythropoietin expression, but there was no correlation with erythropoietin receptor. No correlation was found between Hb and erythropoietin (r = 0.07, P = 0.36) or erythropoietin receptor (r = -0.02, P = 0.8), and no survival difference between high and low erythropoietin or erythropoietin receptor expression (P = 0.59 and P = 0.98, respectively). The mRNA expression of erythropoietin (P = 0.03) but not erythropoietin receptor (P = 0.62) was significantly increased in 11 paired samples of HNSCC. CONCLUSION: In vivo, the HIF pathway regulates erythropoietin at the mRNA level but not erythropoietin receptor expression in HNSCC. Anemia does not seem to influence the hypoxic microenvironment of tumors sufficiently to alter the expression of erythropoietin. The effects of exogenous erythropoietin may be acting via receptors expressed on tumor cells in vivo, or on vascular cells, which also express the pathway.

Anemia↗
PubMed2005

Erythropoietin and erythropoietin receptor expression in human prostate cancer.

Erythropoietin is a hematopoietic cytokine that regulates the production of red blood cells. Erythropoietin is normally produced in the adult kidney in a hypoxia-inducible manner. The recombinant form of human erythropoietin is in clinical use for the prevention and treatment of anemia that is associated with cancer and its treatment with chemoradiation therapy. A series of recent studies from our laboratory and others have reported the expression of receptors for erythropoietin in several different types of human cancer cells. In the present study, we investigated the expression of erythropoietin receptor and its ligand erythropoietin in human prostate cancer. In clinical specimens of prostate cancer, we found abundant expression of erythropoietin receptor protein in all primary tumors examined using immunohistochemistry. Furthermore, we observed erythropoietin coexpression in prostate cancer cells by immunohistochemical analysis. To determine whether monolayer cultures of continuous cell lines derived from prostate cancer also express erythropoietin receptor and erythropoietin, we studied well-characterized hormone-responsive (LNCaP) and hormone-refractory (PC-3) prostate cancer cell lines. We performed reverse-transcription and polymerase chain reaction assays to detect erythropoietin receptor and erythropoietin mRNA transcripts, and also immunoprecipitation and immunoblotting to detect erythropoietin receptor protein expression in prostate cancer cells. These experiments revealed the expression of both erythropoietin receptor and erythropoietin in LNCaP and PC-3 cells suggesting that these prostate cancer cell lines may serve as useful experimental models for further studies of erythropoietin and erythropoietin receptor function in prostate cancer. The coexpression of erythropoietin receptor and its ligand erythropoietin in human prostate cancer cells suggests the potential for growth regulation by erythropoietin-erythropoietin receptor in an autocrine or paracrine manner.

Animals↗
PubMed2001

Decreased serum erythropoietin and its relation to anti-erythropoietin antibodies in anaemia of systemic lupus erythematosus.

OBJECTIVE: This study was performed to assess erythropoietin levels and anti-erythropoietin antibodies in patients with systemic lupus erythematosus (SLE). METHODS: The sera of 100 patients with SLE were investigated for serum erythropoietin levels and the presence of anti-erythropoietin antibodies by ELISA. Routine laboratory parameters such as peripheral blood count, relevant parameters of blood chemistry, and immunological parameters of SLE were recorded. RESULTS: Erythropoietin levels were significantly decreased in SLE patients when related to individual haemoglobin and haematocrit values (P<0.001), suggesting an inadequate erythropoietin response in SLE. Anti-erythropoietin antibodies were found in 46% of SLE patients, and erythropoietin levels (but not haemoglobin or haematocrit values) were significantly decreased in these patients compared with patients without anti-erythropoietin antibodies. Serum erythropoietin concentration as determined by ELISA was reduced in the presence of anti-erythropoietin antibodies. Furthermore, anti-erythropoietin antibodies also correlated with younger age, decreased serum levels of complement factors C3 and C4 and elevated anti-double-stranded DNA antibodies. CONCLUSIONS: We conclude that the anaemia of SLE is characterized by an inadequate erythropoietin response. Anti-erythropoietin antibodies are frequently present in SLE and interfere with the measurement of serum erythropoietin level. However, these antibodies are not associated with increased severity of SLE-associated anaemia.

Adolescent↗
PubMed1990

Association between recombinant human erythropoietin and quality of life and exercise capacity of patients receiving haemodialysis. Canadian Erythropoietin Study Group.

OBJECTIVE: To determine whether recombinant human erythropoietin improves the quality of life and exercise capacity of anaemic patients receiving haemodialysis. DESIGN: A double blind, randomised, placebo controlled study. SETTING: Eight Canadian university haemodialysis centres. PATIENTS: 118 Patients receiving haemodialysis aged 18-75 with haemoglobin concentrations less than 90 g/l, no causes of anaemia other than erythropoietin deficiency, and no other serious diseases. INTERVENTIONS: Patients were randomised to three groups to receive placebo (n = 40), erythropoietin to achieve a haemoglobin concentration of 95-110 g/l (n = 40), or erythropoietin to achieve a haemoglobin concentration of 115-130 g/l (n = 38). Erythropoietin was given intravenously thrice weekly, initially at 100 units/kg/dose. The dose was subsequently adjusted to achieve the target haemoglobin concentration. All patients with a serum ferritin concentration less than 250 micrograms/l received oral or intravenous iron for one month before the study and as necessary throughout the trial. MAIN OUTCOME MEASURES: Scores obtained with kidney disease questionnaire, sickness impact profile, and time trade off technique; and results of six minute walk test and modified Naughton stress test. RESULTS: The mean (SD) haemoglobin concentration at six months was 74 (12) g/l in patients given placebo, 102 (10) g/l in those in the low erythropoietin group, and 117 (17) g/l in those in the high erythropoietin group. Compared with the placebo group, patients treated with erythropoietin had a significant improvement in their scores for fatigue, physical symptoms, relationships, and depression on the kidney disease questionnaire and in the global and physical scores on the sickness impact profile. The distance walked in the stress test increased in the group treated with erythropoietin, but there was no improvement in the six minute walk test, psychosocial scores on the sickness impact profile, or time trade off scores. There was no significant difference in the improvement in quality of life or exercise capacity between the two groups taking erythropoietin. Patients taking erythropoietin had a significantly increased diastolic blood pressure despite an increase in either the dose or number of antihypertensive drugs used. Eleven of 78 patients treated with erythropoietin had their sites of access clotted compared with only one of 40 patients given placebo. CONCLUSIONS: Patients receiving erythropoietin were appreciably less fatigued, complained of less severe physical symptoms, and had moderate improvements in exercise tolerance and depression compared with patients not receiving erythropoietin. At the doses used in this trial there was a higher incidence of hypertension and clotting of the vascular access in patients treated with erythropoietin.

Adolescent↗
PubMed2005

Erythropoietin and erythropoietin receptor expression in head and neck cancer: relationship to tumor hypoxia.

PURPOSE: Erythropoietin, an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor [erythropoietin receptor (EPOR)]. The recombinant form of human erythropoietin is used to prevent or treat anemia in cancer patients. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, erythropoietin treatment was associated with poorer locoregional progression-free survival. The purpose of our study was to determine whether EPOR and its ligand erythropoietin are expressed in primary head and neck cancer. We also investigated the hypothesis that erythropoietin expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness, and poor prognosis. EXPERIMENTAL DESIGN: Twenty-one patients received an i.v. infusion of the hypoxia marker pimonidazole hydrochloride before multiple tumor biopsies. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for EPOR and erythropoietin expression and pimonidazole binding. RESULTS: EPOR expression was present in tumor cells in 97% of the biopsies. Coexpression of erythropoietin was observed in 90% of biopsies. Erythropoietin and pimonidazole adduct staining did not always colocalize within tumors, but there was a significant positive correlation between levels of microregional erythropoietin expression and pimonidazole binding. CONCLUSIONS: The coexpression of erythropoietin and EPOR in tumor cells suggests that erythropoietin may potentially function as an autocrine or paracrine factor in head and neck cancer. The expression of the hypoxia-inducible protein erythropoietin in tumor cells correlates with levels of tumor hypoxia.

Disease-Free Survival↗
PubMed1990

Dynamics of erythropoietin receptor expression on erythropoietin-responsive murine cell lines.

We examined erythropoietin receptor expression in two murine cell lines, B6SUtA and DA-1, that respond to erythropoietin in different ways. While B6SUtA cells undergo erythroid differentiation with limited proliferation after addition of erythropoietin, DA-1 cells show only a proliferative response. Equilibrium binding experiments with 125I-erythropoietin revealed that both B6SUtA and DA-1 cells express a single class of erythropoietin receptors. In the absence of erythropoietin, B6SUtA cells exhibited 145 receptors per cell with a dissociation constant (kd) of 380 pmol/L. Six days after induction with erythropoietin, the B6SUtA cells displayed 310 receptors per cell without a change in binding affinity; exposure to erythropoietin also increased cellular hemoglobin content. DA-1 cells adapted to erythropoietin-dependent growth over a period of months and exhibited a progressive increase in erythropoietin receptor expression, from 85 per cell (kd = 540) to 550 per cell (kd = 530), although the cells remained uniformly benzidine-negative. We interpret the data with B6SUtA cells to indicate that early erythroid differentiation stages are attended by an increase in erythropoietin receptor display, coordinate with the initiation of expression of erythroid-specific genes. In contrast, the results with DA-1 cells are most compatible with clonal selection as the mechanism underlying enhanced receptor expression. Thus, display of the erythropoietin receptor is dynamic and can be modulated during the course of erythropoietin-induced differentiation.

Cell Differentiation↗
PubMed1987

Amniotic fluid erythropoietin correlates with umbilical plasma erythropoietin in normal and abnormal pregnancy.

In the human fetus, elevated plasma erythropoietin levels have been found in high-risk pregnancies at delivery. We examined the relationship of amniotic fluid erythropoietin and umbilical plasma erythropoietin at delivery in 17 normal pregnancies, 41 hypertensive pregnancies, and 37 insulin-treated diabetic pregnancies terminated by elective cesarean section without labor. An additional 27 insulin-treated diabetic patients were studied after undergoing variable durations (86-1184 minutes) of labor. Erythropoietin was analyzed using a highly sensitive and specific radioimmunoassay technique. Fetal plasma erythropoietin concentrations were elevated above the control upper range (50.3 mU/mL) in 59% of the hypertensives and in 38% of the diabetics. The amniotic fluid erythropoietin values were significantly lower than the umbilical plasma erythropoietin values in each study group. Although the umbilical plasma erythropoietin values in the abnormal pregnancy groups differed considerably from the corresponding levels in the controls, the ratio of amniotic fluid erythropoietin to umbilical plasma erythropoietin was approximately the same in controls, hypertensives, and diabetics. Furthermore, the plasma and amniotic fluid levels (In transformed) correlated highly significantly in all three individual groups in absence of labor. In the diabetic labor group, this relationship was nonsignificant. We conclude that in the absence of labor, amniotic fluid erythropoietin reflects fetal plasma erythropoietin. We speculate that amniotic fluid erythropoietin may be an antepartum indicator of fetal hypoxemia.

Amniotic Fluid↗
PubMed1997

Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death.

Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.

Animals↗
PubMed2002

Erythropoietin and erythropoietin-receptor producing cells demonstrated by in situ hybridization in mouse visceral yolk sacs.

We have previously demonstrated that mRNAs for erythropoietin and the erythropoietin receptor temporarily express on the visceral yolk sacs on days 9-11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for erythropoietin or erythropoietin receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while erythropoietin-receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are erythropoietin-receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is erythropoietin-responding cells via both autocrine and paracrine routes.

Animals↗
PubMed1993

Erythropoietin induces the association of phosphatidylinositol 3'-kinase with a tyrosine-phosphorylated protein complex containing the erythropoietin receptor.

Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin-sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated erythropoietin receptor.

Cell Line↗
PubMed1991

Multimeric structure of the membrane erythropoietin receptor of murine erythroleukemia cells (Friend cells). Cross-linking of erythropoietin with the spleen focus-forming virus envelope protein.

In erythroleukemia cells infected with the polycythemia strain of the Friend virus complex, erythropoietin could be cross-linked mainly to a protein of 63 kDa when using disuccinimidyl suberate. In contrast, erythropoietin in other erythroleukemia cells cross-linked to two proteins of 85 and 100 kDa. When native erythropoietin receptor complexes were immunoprecipitated, the 63-kDa erythropoietin-cross-linked protein could be precipitated both by antibodies directed against the intracellular part of the cloned chain of the erythropoietin receptor and by antibodies directed against the envelope proteins of the Friend virus. However, after denaturation of the complexes, the 63-kDa protein was only precipitated by antibodies directed against the envelope proteins of the Friend virus. Enzymatic deglycosylation confirmed that erythropoietin was cross-linked with the envelope protein of the defective virus and bidimensional diagonal gel electrophoresis analyses showed that some of the erythropoietin cross-linked envelope proteins were dimerized by disulfide bonds. Thus, the main erythropoietin-receptor complex in the plasma membrane of these cells consisted of a molecule of the cloned chain of the erythropoietin receptor noncovalently associated with one or two disulfide-bonded molecule(s) of the envelope protein of the defective virus. Moreover, our results also showed that the viral envelope protein associated with the cloned chain of the erythropoietin receptor at a site distinct from the erythropoietin binding site.

Animals↗