EpoR Expression Research Articles

Functional conservation of erythropoietin signaling in zebrafish

Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.

Human recombinant erythropoietin does not promote cancer growth in presence of functional receptors expressed in cancer cells

Human recombinant erythropoietin (hrEPO) therapy might be associated with tumor progression and death. This effect has been suggested to be secondary to rhEPO binding to its receptor (EPOR) expressed on cancer cells. However, there are several concerns about EPOR functionality when expressed on cancer cells. In this paper we have provided evidence that EPOR expressed in cancer cells could be implicated in proliferation events because a transfection of EPOR siRNA to EPOR-expressing bladder cancer cells resulted in a marked reduction in cell growth. However, these cell lines do not grow in the presence of hrEPO. Furthermore, bladder cancer patients that expressed EPOR in tumor samples had a reduced survival in absence of rhEPO treatment. Therefore, EPOR is implicated in bladder cancer growth but this effect appears to be independent from rhEPO supplementation. Reports which suggest that rhEPO promotes cancer growth due to the expression of EPOR in cancer cells must be observed with caution since in the presence of functional EPOR rhEPO does not promote growth.

Expression of erythropoietin and its receptor in neuroblastomas

Children with high-risk neuroblastomas (NB) potentially may benefit from treatment with recombinant human erythropoietin (Epo). Epo is a stimulator of erythropoiesis, acting through its receptor (EpoR). The objective of the current study was to evaluate expression levels of Epo and EpoR in NB and in normal tissues and their effects on the proliferation of tumor cells. A tissue microarray study was performed with 101 primary tumors, 39 paired metastases, 56 paired control tissues, and 6 human NB cell lines. Immunohistochemical staining was performed using antibodies against Epo and EpoR. Immunostaining intensity was evaluated by using a semiquantitative score based on the percentage of positive cells. An in vitro analysis of cell proliferation and cell cycle in the presence of recombinant Epo was performed in the 6 cell lines. The expression of EpoR was found to be significantly higher in tumors than in paired control tissues (P < .0001) compared with the expression of Epo (P = .06), and the expression of EpoR was significantly higher in lymph node metastases than in paired primary tumors (P = .02) compared with Epo (P = .99). Survival analysis demonstrated that the patients who had tumors with the highest expression of EpoR had a significantly better overall survival (P = .03). In the in vitro study, recombinant Epo did not modify proliferation and cell cycle in the cell lines regardless of the EpoR expression level. Epo and EpoR were expressed in NB but did not modify tumor cell proliferation. The results of the current study suggested that Epo may be used safely for supportive care in children with NB.

Endogenous Erythropoietin Signaling Is Required for Normal Neural Progenitor Cell Proliferation

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

Expression and function of erythropoietin receptors in tumors

Safety concerns surrounding the use of recombinant human erythropoietin (Epo) to treat anemia in cancer patients were raised after 2 recent clinical studies reported a worse survival outcome in patients who received epoetin alpha or epoetin beta compared with patients who received placebo. Although those findings contrasted with previous clinical studies, which demonstrated no difference in survival for cancer patients who received erythropoiesis-stimulating agents (ESAs), some investigators have suggested a potential role for ESAs in promoting tumor growth through 1) stimulation of Epo receptors (EpoR) expressed in tumors, 2) stimulation and formation of tumor vessels, and/or 3) enhanced tumor oxygenation. The first and second hypotheses appeared to be supported by some EpoR expression and ESA in vitro studies. However, these conclusions have been challenged because of poor specificity of EpoR-detection methodologies, conflicting data from different groups, and the lack of correlation between in vitro data and in vivo findings in animal tumor models. For this report, the authors reviewed the biology of EpoR in erythropoiesis and compared and contrasted the reported findings on the role of ESAs and EpoR in tumors.

Erythropoietin and Erythropoietin Receptor Expression in Vestibular Schwannoma

Hypoxia-inducible factor (HIF)-1alpha, erythropoietin (Epo), Epo receptor (EpoR), and bcl-2 are expressed in both sporadic unilateral vestibular schwannomas (VSs) and those associated with neurofibromatosis Type 2, and the expression data correlate with clinicopathological tumor features including microvessel density and Ki-67-labeling index. Erythropoietin expression is regulated by the transcription factor, HIF-1alpha. Erythropoietin signaling via EpoR results in stimulation of cell proliferation and elevated expression of the antiapoptotic protein, bcl-2, and then inhibition of apoptosis. Erythropoietin has been shown to be associated with Schwann cell proliferation, and a recent report suggested a role in VS growth. Immunohistochemical analysis of HIF-1alpha, Epo, EpoR, and bcl-2 was performed on formalin-fixed paraffin-embedded archival surgical specimens. Microvessel density and Ki-67-labeling index of VS were analyzed and correlated with the immunoreactivity pattern of the examined factors. Immunoreactivity data demonstrate robust protein expression for HIF-1alpha, Epo, EpoR, and bcl-2 in VS. Sixty-six percent of the cases showed Epo expression, and EpoR was found in 85% of tumor samples. A significantly positive correlation of the immunoreactivity scores of Epo/EpoR and bcl-2 expression could be noted. In case of tumor specimens with high levels of HIF-1alpha expression, a significantly higher Ki-67-labeling index was observed. There was no correlation between the expression of HIF-1alpha, Epo, EpoR, and bcl-2 and microvessel density, tumor size, sex, and age. Expression of Epo and EpoR might suggest a functional role in VS biology. The observed correlation of Epo/EpoR and bcl-2 expression levels may suggest a proliferative and antiapoptotic role of the Epo/EpoR system in VS.

Lovastatin suppresses erythropoietin receptor surface expression through dual inhibition of glycosylation and geranylgeranylation

Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64 kDa and the mature 66 kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and geranylgeranylation.

Brain erythropoietin receptor expression in Alzheimer disease and mild cognitive impairment.

Cellular mechanisms conferring neuroprotection in the brains of patients with Alzheimer disease (AD) remain incompletely understood. Erythropoietin (Epo) and the erythropoietin receptor (EpoR) are expressed in neural tissues and protect against oxidative and other stressors in various models of brain injury and disease. Our objective in this study was to determine whether EpoR is upregulated in the brains of persons with sporadic AD and mild cognitive impairment (MCI). Postmortem hippocampus and temporal cortex from subjects with AD, MCI, and no cognitive impairment (NCI) were procured from the Religious Orders Study. Total immunoreactive EpoR protein was determined by Western blotting. Astrocytes expressing immunoreactive EpoR were quantified in 4 temporal and 6 hippocampal regions, and correlated with clinical, neuropsychologic, and neuropathologic indices. Total immunoreactive EpoR protein was markedly increased in AD and MCI temporal cortex versus NCI tissues. Composite measures of glial EpoR expression in temporal cortex layers I to IV were significantly greater in the MCI group compared with the NCI and AD groups. Hippocampal EpoR scores were increased in persons with MCI and AD relative to those with NCI. There was substantial subregional heterogeneity in disease-related EpoR expression patterns in AD and MCI temporal cortex and hippocampus. There was no association of EpoR-positive astrocytes with summary measures of global cognition or AD pathology. We conclude that upregulation of EpoR in temporal cortical and hippocampal astrocytes is an early, potentially neuroprotective, event in the pathogenesis of sporadic AD.

Increasing expression of hypoxia-inducible proteins in the Barrett's metaplasia–dysplasia–adenocarcinoma sequence

Hypoxia-associated markers are involved in the progression of several malignancies, but are relatively unstudied in Barrett's carcinogenesis. Our aim was to assess the immunohistochemical expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, erythropoietin (Epo), Epo receptor (Epo-R), Glut-1 and vascular endothelial growth factor (VEGF) along with Ki67/MIB-1 in the Barrett's metaplasia–dysplasia–adenocarcinoma sequence. Endoscopic biopsies of normal squamous epithelium (NSE) (n=20), columnar-lined oesophagus (CLO) (n=15), CLO with intestinal metaplasia (n=20), dysplasia (n=17) and Barrett's type adenocarcinoma (n=20) were obtained. Immunohistochemistry was performed on the paraffin-embedded tissue. A score was calculated for each marker (range 0−300) by multiplying intensity (none 0, weak 1, moderate 2, strong 3) by percentage of expression (range 0–100). Significant increases in the expression of HIF-2α (P=0.014), VEGF (P<0.0001), Epo-R (P<0.0001) and Ki67 (P<0.0001) were found as tissue progressed from NSE to adenocarcinoma. HIF-2α was expressed late in the sequence and was only seen in dysplasia and adenocarcinoma. High HIF-2α expression was seen in 12 out of 20 Barrett's type adenocarcinoma. The late expression of HIF-2α in the Barrett's carcinogenesis sequence and its high expression in adenocarcinoma suggest that it is worth further investigation as a marker of disease progression and therapeutic target.

Erythropoietin is neuroprotective on GABAergic neurons against kainic acid-excitotoxicity in the rat spinal cell cultures

The aim of this study was to investigate whether erythropoietin (EPO) protect the spinal GABAergic neurons against kainic acid (KA)-excitotoxic damage in rat spinal cord cell cultures. We performed immunohistochemical staining and Western blotting of glutamate decarboxylase 67 (GAD67), one isoform of GABA-producing enzyme, which was considered to have involved in nonsynaptic functions, such as energy metabolism or trophic support. T exposure to KA significantly reduced the intensity of GAD67 expression in the GABAergic neurons and whole cell lysate, indicating that the excitotoxic damage on the GABAergic neurons may lead to reduction of the GAD67 production following KA-exposure. We found that post-treatment of EPO for 48 h after KA-injury remarkably enhanced the expression level of GAD67 and erythropoietin receptors (EpoR), which were deteriorated by KA. Our confocal images clearly demonstrated a remarkably enhanced expression of EpoR on the surface of the GABAergic neurons by post-treated EPO after KA-damage. This result suggests that the neuroprotective effect of post-treated EPO on the GABAergic neurons can be related to the EPO-mediated EpoR upregulation following KA-excitotoxicity. We observed that the post-applied EPO clearly increased expression of tyrosine Janus kinase 2 (JAK2), which is known to be the first step of EpoR-stimulation. In conclusion, the post-treated EPO is a potent protector of the spinal GABAergic neurons against KA-excitotoxicity and regulates production of GAD67 for the multiple trophic roles after KA-induced disturbance. We suggested that the protective effect of post-treated EPO on the GABAergic neurons is mediated by signal transduction involving EpoR-dependent JAK2 pathway.

Erythropoietin/erythropoietin‐receptor system is involved in angiogenesis in human hepatocellular carcinoma

Ribatti D, Marzullo A, Gentile A, Longo V, Nico B, Vacca A & Dammacco F (2007) Histopathology 50, 591–596 Erythropoietin/erythropoietin-receptor system is involved in angiogenesis in human hepatocellular carcinomaAimsTo correlate microvascular density and erythropoietin (Epo)/Epo-receptor (EpoR) expression in endothelial and tumour cells with histopathological type in hepatocellular carcinoma (HCC).Methods and resultsSpecimens of primary HCC obtained from 50 patients who had undergone curative hepatectomy were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. Poorly differentiated HCC had a higher degree of vascularization than other stages and Epo/EpoR expression in both tumour and endothelial cells increased in parallel with grade of malignancy and was highly correlated with the extent of angiogenesis.ConclusionsEpo/EpoR levels correlate with angiogenesis and progression of patients with HCC and these findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. It is thus suggested that Epo is an important factor in hepatic tumour angiogenesis. Understanding the mechanisms of HCC angiogenesis provides a basis for a rational approach to the development of antiangiogenic therapy in patients with hepatic cancer.

Erythropoietin/erythropoietin receptor system is involved in angiogenesis in human neuroblastoma

Ribatti D, Poliani P L, Longo V, Mangieri D, Nico B & Vacca A (2007) Histopathology50, 636–641 Erythropoietin/erythropoietin receptor system is involved in angiogenesis in human neuroblastomaAimsPrevious studies have shown that increased vascularity is associated with tumour progression in human neuroblastoma (NB). The involvement of erythropoietin (Epo) in tumour angiogenesis has also been reported. The aim of this study was to correlate microvascular density and Epo/Epo-receptor (EpoR) expression in endothelial and tumour cells to the clinical stage of NB.Methods and resultsSpecimens of NB obtained from 20 patients were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. The extent of angiogenesis was found to be up-regulated in advanced disease. In keeping with this observation, Epo/EpoR expression in tumour and endothelial cells, respectively, was also highly correlated with the extent of angiogenesis and higher clinical stage.ConclusionsThe correlation of Epo/EpoR expression with angiogenesis and tumour progression suggests the presence of a loop in the Epo–EpoR system. Epo is secreted by tumour cells and affects vascular endothelial cells via its receptor, promoting tumour angiogenesis in a paracrine manner. Data suggest that Epo represents an important mediator in NB angiogenesis. Understanding the mechanisms of NB angiogenesis provides the basis for a rational approach to the development of antiangiogenic therapy in patients affected by NB.

Post‐pneumonectomy (PNX) lung expansion enhances expression of erythropoietin receptor (EPO‐R) through an Akt‐associated pathway

To examine if the elevated EPO-R protein level post-PNX (AJP 287:, 2004) is related to lung expansion, we replaced the right lung in litter-matched adult foxhounds (22.2±4.0kg) by a custom-shaped silicone prosthesis inflated with SF6/air to prevent mediastinal shift and minimize expansion of the remaining lung. Normal (pre-PNX) lung tissue was sampled from the removed right upper lobe. Three wk post-PNX, the prosthesis was either acutely deflated (DEF, n=3) to impose lung expansion separate from other post-PNX stimuli, or kept inflated (INF, n=3). Three days later, lung tissue was sampled from the remaining left upper lobe and analyzed by real-time PCR (EPO-R mRNA) and immunoblots (EPO-R, total and phospho-Akt) normalized to cyclophilin and β-actin, respectively. Triplicate assays used separate tissue samples. Results are shown as ratios to pre-PNX (±SD). ∗p<0.05 vs. 1.0, paired t-test. In the INF group, no significant post-PNX change was seen. In the DEF group, EPO-R mRNA and protein were 183% and 71% higher and p-Akt/total-Akt 124% higher than pre-PNX. Results suggest that post-PNX lung expansion enhances EPO-R expression through an Akt-associated pathway. Supported by NHLBI RO1 HL40070, 62873, 45716, 54060 and R01-DK48482, DK-20543 and the Simmons Family Foundation.

Expression of Erythropoietin and Erythropoietin Receptor in Cervical Cancer and Relationship to Survival, Hypoxia, and Apoptosis

Physiologically, hypoxia induces the expression of erythropoietin (Epo) in adult kidney cells. Epo, in turn, acts on the Epo receptor (EpoR) in RBC precursors to stimulate growth and prevent apoptosis. Because hypoxia plays a major role in the malignant progression of tumors and Epo and its receptors have also been detected in malignant tumors, we investigated the expression of Epo and EpoR and their relationship with hypoxia, proliferation, apoptosis, and clinicopathologic variables in cervical cancer. Intratumoral oxygen measurement and needle biopsies of the tumors were done in 48 patients with cervical cancer. The obtained tissue was analyzed by immunohistochemistry with antibodies against Epo, EpoR, and Ki-67 as well as by terminal deoxynucleotidyl transferase-mediated deoxyuracil triphosphate nick-end labeling assays. Epo and EpoR were expressed in 88% and 92% of samples, respectively. Cervical cancers with higher Epo expression showed a significantly reduced overall survival (3 years, 50.0% versus 80.6%; P = 0.0084). Epo and EpoR expression correlated significantly with apoptosis (r = 0.49, P = 0.001 and r = 0.36, P = 0.021). Furthermore, EpoR expression correlated significantly with tumor size (r = 0.32, P = 0.032) and was significantly associated with the presence of lymphovascular space involvement (P = 0.037). However, we observed no correlation between Epo or EpoR expression and intratumoral hypoxia, although in well-oxygenated tumors, EpoR localized significantly more often to the invasion front (P = 0.047). This study analyzes Epo/EpoR expression and their relationship with intratumoral pO(2) levels as well as with survival in patients with cervical cancer. The data suggest a critical role of the endogenous Epo/EpoR system in cervical cancer.

The Wilms' Tumor Suppressor Wt1 Activates Transcription of the Erythropoietin-Receptor in Hematopoietic Progenitor Cells.

The Wilms' tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1 deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+ cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoR was significantly reduced in hematopoietic progenitor cells that lack Wt1 compared to Wt1+/+ cells. The decrease of EpoR mRNA was not due to a general down-regulation of gene expression in Wt1−/− CD117-positive cells, since levels of c-kit and c-mpl expression were normal. In the heart of wild-type and Wt1−/− embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoR und Wt1, no difference in Epo-R expression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoR transcripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoR promoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoR promoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoR gene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoR gene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoR expression in non-erythoid tissues, such as the heart.

Proliferative Defect but Normal Transcriptional Response to Erythropoietin in Diamond Blackfan Anemia Erythroid Progenitor Cells in 2-Phase Erythroid Culture.

Diamond Blackfan anaemia (DBA) is a rare congenital red cell aplasia, characterised by a steroid responsive intrinsic erythropoietic failure. Mutations affecting RPS19 are found in up to 25% of DBA, but the pathophysiological link between ribosomal gene haploinsufficiency and the specific erythroid phenotype of DBA remains elusive. One potential mechanism is that ribosomal insufficiency might affect the level of expression of critical erythroid proteins, resulting in a block in terminal erythroid differentiation and expansion. The aim of this study was to elucidate the cellular and molecular events occurring in response to erythropoietin (EPO) in DBA. We applied a 2-phase erythroid culture system in which we have previously demonstrated a consistent and profound failure of erythroid expansion in DBA cultures, localised to early in the second EPO-dependent phase of culture. The timing of the onset of proliferation in response to EPO was studied by tetrazolium dye assay of enriched erythroid progenitor cells in phase II of culture. An increase in cell number was detectable following 72hrs of EPO exposure in DBA (n=5) as well as control cultures, but with a subsequently slower proliferation rate in DBA. The proliferation rate of both DBA and control cells was enhanced by the addition of dexamethasone. Transcriptional profiles of control and DBA erythroid progenitors were then compared by microarray analysis after 24hrs exposure to EPO. Cy3/Cy5 labelled aRNA was competitively hybridised on an in-house array containing 395 transcript exon identifiers in ENSEMBL. The results showed little difference in expression between control and DBA after 24hrs of EPO exposure, the only significant changes being in the expression of just 4 of the 251 genes expressed above background (CDKN2D, TNFRSF6, MAPKK and CXCR), which were upregulated in DBA (p<0.0001 on t-test analysis). The gene expression profile from 0–96 hrs following EPO exposure was then studied in control and DBA (n=3) erythroid progenitors by real time PCR. There was little change in the first 48hr of exposure to EPO. At 72hrs the expression of both EPOR and α-GLOBIN was increased 3–6 fold in both DBA and controls in comparison with pre-EPO, confirming the occurrence of an erythroid-specific transcriptional response to EPO in DBA erythroid progenitors. These results are consistent with a qualitatively normal onset of terminal erythroid maturation in DBA, rather than a block in differentiation, and tend to favour an alternative hypothesis that the erythroid failure in DBA is the result of ribosomal insufficiency at the simultaneous onset of both rapid cell proliferation and a high rate of globin synthesis. In keeping with this, the expression of RPS19 showed 1.5–2 fold increase at 72hrs after EPO exposure, with 3.5 fold increase in the presence of dexamethasone, with equivalent results in both DBA and controls. We now aim to study translational and cell cycle events during this important wave of erythroid proliferation to further elucidate the molecular basis for the specific erythroid defect in DBA, despite the ubiquitous cellular requirement for ribosomal biogenesis.

The Membrane-proximal Region of the Thrombopoietin Receptor Confers Its High Surface Expression by JAK2-dependent and -independent Mechanisms

Janus tyrosine kinase 2 (JAK2) is essential for signaling by the thrombopoietin (TpoR) and erythropoietin (EpoR) receptors. In the absence of JAK2 most EpoR molecules are retained in the endoplasmic reticulum in an Endo H-sensitive form. In contrast, we show that in the absence of JAK2 a large fraction of the TpoR is processed to the mature Endo H-resistant form and reaches the cell surface. By studying chimeras of the TpoR and EpoR we show that high surface expression of the TpoR is entirely conferred by the membrane-proximal region of the intracellular domain that includes the juxtamembrane, Box 1, and Box 2 regions. The TpoR intracellular domain shows similar effects on receptor endocytosis rate as that of the EpoR, but does stabilize the mature receptor isoform from degradation. Co-expression of JAK2 further stabilizes mature TpoR and thus further increases its surface expression. This JAK2 effect depends on the Box 1 region, the only JAK2 interacting site in the TpoR. By contrast, EpoR requires Box 1 as well as the flanking 20 residues on the C-terminal side for JAK2 interaction and JAK2-dependent surface expression. Our study suggests that whereas cell surface expression of type I cytokine receptors requires their cognate JAKs, the mechanisms governing receptor-JAK interactions differ among receptors interacting with the same JAK protein.

A “classical” homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells

The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule (“site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common β chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A “site 2” Epo mutant did not, and we could not detect expression of the common β chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits, comprising the “classical” Epo receptor signaling complex.

Erythropoietin activates the phosphoinositide 3-kinase/Akt pathway in human melanoma cells

Erythropoietin (Epo) is used commonly to treat cancer and/or therapy-related anemia. Until recently, Epo was considered to be a specific stimulator of erythropoiesis, acting via its receptor, EpoR. It becomes clear, however, that EpoR is expressed in a variety of cell types other than hematopoietic cells, and that Epo is a potent cytoprotective cytokine increasing cell survival under hypoxic conditions. Epo and EpoR are also expressed in various malignant tumors, and EpoR expression shows association with tumor invasion and progression. Recently, a functional Epo autocrine signaling mechanism was also detected in human melanoma cells. In this study, we examined the hypothesis that Epo activates the Akt signaling pathway in human melanoma cells and thus promotes the survival of tumor cells. The Akt signaling pathway in response to Epo was examined in melanoma. Similar to Epo, the expression of EpoR was up-regulated in response to hypoxia and Epo stimulation in melanoma cells. Melanoma cells constitutively expressed Akt with variable expression of mammalian target of rapamycin, and Epo dose-dependently induced their activity. Epo increased Akt kinase activity, which was abrogated by co-treatment with LY294002, a specific blocker of phosphoinositide 3-kinase. LY294002 also inhibited the cytoprotective effects of Epo in melanoma cells under both normoxic and hypoxic conditions. Our results suggest that Epo promotes melanoma cell survival by activating an Akt-dependent signaling pathway.

Expression of erythropoietin and its receptor in neuroblastomas

9054 Background: Children with neuroblastomas (NB) may potentially benefit from treatment with recombinant human erythropoietin (Epo). Epo is a specific stimulator of erythropoiesis, acting via its specific receptor (EpoR). The aim of this study was to evaluate the expression of Epo and EpoR in NB and in normal tissues and their action in the proliferation of tumor cells. Methods: A tissue microarrays study was performed on 101 patients with NB from 3 hospitals (16 stage I; 7, II; 22, III; 48, IV and 8, IVS). Four blocks were constructed and contained 101 primary tumors, 39 paired metastases (35 lymph nodes), 56 paired control normal tissues and 6 cell lines of NB. Immunohistochemical staining was performed on sections using antibodies against Epo and EpoR. Immunostaining intensity was evaluated by a semi-quantitative score based on the percentage of positive cells. The wilcoxon signed rank test was applied for the comparison of paired data. For overall survival analysis, the expression was dichotomized at the median value and cox regression models were used stratified by hospital. An in vitro study of cell proliferation in presence of recombinant Epo was carried out with two of the 6 cell lines, one expressing EpoR, the other not. Results: Median follow-up was 64 months. The expression of EpoR was significantly higher in tumors than in paired control tissues (p&lt;0.0001) but not for Epo (p=0.06). When restricted to the lymph nodes metastases, the level of expression of EpoR was significantly more expressed in the metastases than in primary tumors (p=0.02), contrary to Epo (p=0.99). Survival analysis showed that patients who expressed more EpoR had a statistically significant better overall survival than those who did not (p=0.03). Furthermore, the expression of EpoR was an independent prognostic factor from induction chemotherapy, tumor stage and age (p=0.02). No significant differences were found in Epo analyses. EpoR was expressed in only three cell lines and Epo not in any of them. In the in vitro study, recombinant Epo did not modify the proliferation of both cell lines. Conclusions: Epo and EpoR were expressed in NB but did not modify cells proliferation. The use of Epo in children with NB could be proposed but its benefit should be confirmed in a clinical trials. No significant financial relationships to disclose.