Erythropoietin Receptor mRNA Expression Research Articles

\u0397\u0399F1\u03b1, EGR1 and SP1 co-regulate the erythropoietin receptor expression under hypoxia: an essential role in the growth of non-small cell lung cancer cells

BackgroundOverexpression of erythropoietin (EPO) and EPO receptor (EPO-R) is associated with poor prognosis in non-small-cell lung carcinoma (NSCLC). Hypoxia, a potent EPO inducer, is a major stimulating factor in the growth of solid tumors. However, how EPO-R expression is regulated under hypoxia is largely unknown.MethodsThe role of EPO-R in NSCLC cell proliferation was assessed by RNA interference in vitro. Luciferase reporter assays were performed to map the promoter elements involved in the EPO-R mRNA transcription. Nuclear co-immunoprecipitation and chromatin immunoprecipitation were performed to assess the interaction among transcription factors HIF1α, SP1, and EGR1 in the regulation of EPO-R under hypoxia. The expression of key EPO-R transcription factors in clinical specimens were determined by immunohistochemistry.ResultsHypoxia induced a dosage and time dependent EPO-R mRNA expression in NSCLC cells. Knockdown of EPO-R reduced NSCLC cell growth under hypoxia (P < 0.05). Mechanistically, a SP1-EGR1 overlapped DNA binding sequence was essential to the hypoxia induced EPO-R transcription. In the early phase of hypoxia, HIF1α interacted with EGR1 that negatively regulated EPO-R. With the exit of EGR1 in late phase, HIF1α positively regulated EPO-R expression through additive interaction with SP1. In clinical NSCLC specimen, SP1 was positively while EGR1 was negatively associated with active EPO-R expression (P < 0.05).ConclusionsHIF1α, SP1 and EGR1 mediated EPO-R expression played an essential role in hypoxia-induced NSCLC cell proliferation. Our study presents a novel mechanism of EPO-R regulation in the tumor cells, which may provide information support for NSCLC diagnosis and treatment.Graphical abstract

Erythropoietin signal protected human umbilical vein endothelial cells from high glucose‐induced injury

High glucose (HG) induces endothelial injury in vasculature, leading to tissue injury in diabetic condition. Therefore, diabetes is one of the major cause of end-stage kidney disease as well as cardiovascular diseases. Chronic inflammation is involved in the progression of HG-induced cell injury. Recently, it has been reported that erythropoietin (EPO) protects the tissues from some kind of injury, such as hypoxia and mechanical stress. However, the contribution of EPO to HG-induced tissue injury remains to be explored. Therefore, we hypothesized that EPO protects endothelial cells from HG-induced injury via the regulation of inflammatory and anti-inflammatory balance. We performed genome-wide transcriptome profiling in human umbilical vein endothelial cells (HUVEC), which were stimulated by HG with/without EPO treatment and detected the expression of inflammation associated genes. The expression pattern of mRNA expression in HG stimulated HUVEC with/without EPO were different in hieralchial clustering analysis. While inflammatory cytokines/chemokines mRNA expression were increased by the HG stimulation in HUVEC, Th2-related cytokine receptors and intracellular signaling molecules showed the reduced mRNA expression levels. EPO treatment reduced inflammatory cytokines/chemokines mRNA expression and increased Th2-related cytokine mRNA expression levels. Moreover, EPO stimulation increased mRNA expression of EPO receptor and β-common receptor. EPO signaling protects HG-induced cell injury by the regulation of immune balance.

Venlafaxine Mitigates Depressive-Like Behavior in Ovariectomized Rats by Activating the EPO/EPOR/JAK2 Signaling Pathway and Increasing the Serum Estradiol Level.

Reduced estradiol levels are associated with depression in women during the transition to and after menopause. A considerable number of studies focusing on the theme of treating depression through the activation of erythropoietin (EPO)-induced signaling pathways have been published. Venlafaxine is an approved antidepressant drug that inhibits both serotonin and norepinephrine transporters. The aim of the present study was to investigate the effects of venlafaxine on the depressive-like behaviors and serum estradiol levels in female rats following ovariectomy (OVX) and the possible roles of EPO-induced signaling pathways. Venlafaxine (10mg/kg/day) was orally administered to OVX rats over a period of 4weeks using two different treatment regimens: either starting 24h or 2weeks after OVX. Venlafaxine showed a superior efficacy in inducing antidepressant-like effects after an acute treatment (24h post-OVX) than after the delayed treatment (2weeks post-OVX) and was characterized by a decreased immobility time in the forced swimming test. In parallel, venlafaxine induced EPO and EPO receptor mRNA expression and increased levels of phospho-Janus kinase 2 (p-JAK2), phospho-signal transducer and activator of transcription 5, and phospho-extracellular signal-regulated kinase 1/2 in the hippocampus of OVX rats. Meanwhile, rats exhibited a marked reduction in the hippocampal Bax/Bcl2 ratio, caspase-3 activity, and tumor necrosis factor alpha levels after venlafaxine treatment. Venlafaxine also increased the hippocampal brain-derived neurotrophic factor and serum estradiol levels. Based on these findings, venlafaxine exerts a neuroprotective effect on OVX rats that is at least partially attributed to the activation of EPO/EPOR/JAK2 signaling pathways, anti-apoptotic activities, anti-inflammatory activities, and neurotrophic activities, as well as an increase in serum estradiol level. Graphical Abstract ᅟ.

EPOR mRNA level: A valuable prognostic indicator for patients with ER+ breast cancer

Objective: To explore the expression level and prognostic significance of erythropoietin receptor (EPOR) in patients with breast cancer (BRCA) based on estrogen receptor (ER) status and different molecular subtypes. Methods: The Cancer Genome Atlas (TCGA) and GTEx data were collected in GEPIA initially to identify the dysregulated genes. Further, bc- GenExMiner 4.1 online bioinformatics tool was used to evaluate EPOR mRNA differential expression level according to different classification of clinicopathologic parameters in patients with breast cancer. Additionally, the prognostic value between EPOR mRNA expression and free survival of metastatic relapse (MR) or any event (AE, namely any relapse or death) in patients with breast cancer was done. Results: EPOR mRNA was significantly downregulated in BRCA (1 085 cases) compared to normal tissues (291 cases) (P<0.05). Univariate Cox analysis revealed that high EPOR mRNA expression was remarkably correlated to a decreased risk of MR (HR: 0.79, P<0.000 1) and AE (HR: 0.87, P =0.000 7) especially in breast cancer patients with ER+. Besides, high EPOR mRNA level also associated with a favorable MR- free survival (HR: 0.81, P =0.007 2) and AE-free survival (HR: 0.88, P =0.029 9) in ER+ breast cancer patients. However, no similar above phenomenon was detected in ER- patients. Moreover, the subsequent prognostic adjusted analyses and univariate Cox analysis of AE based on SSP or SCM molecular subtypes validated the above results. Conclusion: EPOR mRNA level is a valuable prognostic indicator for patients with ER+ breast cancer.

Significance of anti-EPO receptor antibody in immune-related pancytopenia

Objective: To confirm the presence of erythropoietin receptor (EPOR) antibody in patients with immune-related pancytopenia (IRP) and to evaluate the significance of EPOR in IRP. Methods: A total of 59 newly-diagnosed IRP patients, 62 patients with IRP in remission, 14 patients with aplastic anemia (AA), 15 patients with myelodysplastic syndromes (MDS) and 33 healthy controls were enrolled in this study from January 2013 to June 2015 in Tianjin Medical University General Hospital. The anti-EPOR antibody was detected by enzyme-linked immunosorbent assay(ELISA). The expression of EPOR mRNA was detected by quantitative real-time PCR (qRT-PCR). The correlation between the data and the clinical indicators of patients was analyzed. Results: The levels of anti-EPOR antibodies were higher in the newly-diagnosed IRP patients than in the remission IRP group, AA group, MDS group and controls(0.84±0.39 vs 0.46±0.25, 0.49±0.25, 0.50±0.25, 0.53±0.14, all P<0.05). The expression of EPOR- mRNA was up-regulated in the newly-diagnosed IRP group than in the remission IRP group and the controls. The positive rate of EPOR antibody was significantly elevated in the patients with low hemoglobin level (<100 g/L), positive GlycoA antibody, or low complement C3 (all P<0.05). Anti-EPOR antibody was significantly decreased in the IRP patients responding to immunosuppressive therapy. Conclusions: Anti-EPOR autoantibody is present in patients with IRP. Detection of anti-EPOR antibody has important clinical value in the diagnosis, differential diagnosis and treatment effect evaluation of IRP.

The Local HIF-2α/EPO Pathway in the Bone Marrow is Associated with Excessive Erythrocytosis and the Increase in Bone Marrow Microvessel Density in Chronic Mountain Sickness.

Chronic mountain sickness (CMS) is characterized by excessive erythrocytosis, and angiogenesis may be involved in the pathogenesis of this disease. The bone marrow niche is the primary site of erythropoiesis and angiogenesis. This study was aimed at investigating the associations of the levels of hypoxia-inducible factors (HIFs), erythropoietin (EPO), and erythropoietin receptor (EPOR), as well as microvessel density (MVD) in the bone marrow with CMS. A total of 34 patients with CMS and 30 control subjects residing in areas at altitudes of 3000-4500 m were recruited for this study. The mRNA and protein expression of HIF-2α and EPO in the bone marrow cells was significantly higher in the CMS patients than in the controls. Moreover, changes in HIF-2α expression in CMS patients were significantly correlated with EPO and hemoglobin levels. In contrast, the expression of mRNA and protein expression of HIF-1α and EPOR did not differ significantly between the CMS and control patients. Increased MVD was observed in the bone marrow of the patients with CMS and it was significantly correlated with hemoglobin. Bone marrow cells of CMS patients may show enhanced activity of the HIF-2α/EPO pathway, and EPO may regulate the erythropoiesis and vasculogenesis through autocrine or/and paracrine mechanisms in the bone marrow niche. The increased MVD in the bone marrow of CMS patients appears to be involved in the pathogenesis of this disease.

Low Oxygen Tension Primes Aortic Endothelial Cells to the Reparative Effect of Tissue-Protective Cytokines.

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared with 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast, βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis.

Hepatoprotective effects of erythropoietin on D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice.

Fulminant hepatic failure is a severe clinical syndrome associated with a high rate of patient mortality. Recent studies have shown that in addition to its hematopoietic effect, erythropoietin (EPO) has multiple protective effects and exhibits antiapoptotic, antioxidant and anti-inflammatory activities. The present study aimed to determine the hepatoprotective effect of EPO and to elucidate the underlying mechanisms using a D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced model of acute liver injury. Experimental groups of mice were administered with various doses of EPO (1,000, 3,000 or 10,000 U/kg, intraperitoneal) once per day for 3 days, prior to injection with D-GalN (700 mg/kg)/LPS (10 µg/kg). Mice were sacrificed 8 h after treatment with D‑GalN/LPS. Liver function and histopathology, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH‑Px) activities and EPO receptor (EPOR) and phosphatidylinositol 3-kinase (PI3K) mRNA expression were evaluated. D-GalN/LPS administration markedly induced liver injury, as evidenced by elevated levels of serum aminotransferases, as well as histopathological changes. Compared with the D-GalN/LPS group, pretreatment with EPO significantly decreased the levels of aspartate aminotransferase, alanine aminotransferase and MDA, and increased the activities of SOD and GSH-Px. Furthermore, the protective effects of EPO were paralleled by an upregulation in the mRNA expression of EPOR and PI3K. These data suggest that EPO can ameliorate D-GalN/LPS-induced acute liver injury by reducing oxidative stress and upregulating the mRNA expression of EPOR and PI3K.

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10–50μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2kb 5′. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions.

Expression of erythropoietin receptor and its relation with neovascularization in gliomas

Objective To investigate the expression of erythropoietin receptor (EPOR) in human gliomas and its relationship with histopathological degree and neovascularization.Methods Immunohistochemistry of the tissue microarray was used to detect the expression of EPOR and CD105 in gliomas.Tissue samples included 152 gliomas of different WHO grades and 20 normal brain tissue specimens.Twenty-one gliomas and 6 normal brain tissue specimens were detected by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).Results The expression of EPOR was significantly higher in glioma specimens than in normal brain tissues (x2 =7.7038,P ＜ 0.01),and EPOR expression was positively correlated with the stage of the tumor (rx =0.3151,P ＜ 0.01).CD105 counts of EPOR (艹,(艹)) were greater than those of EPOR(－,+) (P ＜0.05).There was no significant difference in the EPOR mRNA expression in glioma group and normal brain group.Conclusion Tbe expression of EPOR in human glioma exhibits a positive correlation with histopathological degree and neovascularization.There is no up-regulation of EPOR mRNA. Key words: Glioma; Erythropoietin receptors; CD105 ; Tissue arrayer

Erythropoietin receptor expression and its relationship with trastuzumab response and resistance in HER2-positive breast cancer cells

Resistance to trastuzumab is a major issue in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Several potential resistance mechanisms have been investigated, but the results are controversial and no conclusion has been reached. Erythropoietin receptor (EPOR) may function in cell growth, and expressed in various cancer cells. Because the downstream signaling pathways for EPOR and HER2 partially overlapped, we hypothesized that EPOR may play a role in the inhibition effect of trastuzumab and resistance to trastuzumab. Here, we detected the expression of EPOR mRNA and protein in HER2-positive breast cancer cell lines and tissues. EPOR expressed in SKBR3, MDA-MB-453, and UACC-812 cell lines, but not in BT474. Of the 55 HER2-positive cancer tissues, EPOR was positive in 42 samples and highly expressed (H-score ≥ 25) in 24 by immunohistochemistry. The difference between EPOR expression and Ki67 index was significant (P = 0.033), and EPOR expression also positively correlated with higher pathological stage (Spearman correlation coefficient = 0.359; P = 0.007). Exogenous EPO antagonized trastuzumab-induced inhibition of cell proliferation in HER2/EPOR dual-positive breast cancer cells. We then exposed SKBR3 cells to trastuzumab for 4 months to obtain trastuzumab-resistant SKBR3 cell line, which demonstrated higher phosphorylated EPOR level, higher EPO expression and more extracellular secretion than non-resistant parental SKBR3 cells. Downregulation EPOR expression using short hairpin RNA resensitized trastuzumab-resistant cells to this drug, and SKBR3 cells with EPOR downregulation demonstrated attenuated trastuzumab resistance after the same resistance induction. EPOR downregulation plus trastuzumab produced a synergetic action in the inhibition of cell proliferation and invasion in SKBR3 and MDA-MB-453 cell lines. Therefore, EPOR expression may be involved in tumor progression and proliferation in HER2-positive breast cancer. EPO/EPOR contributes to the mechanism of trastuzumab resistance in SKBR3 cell lines, and EPOR downregulation can reverse the resistance to trastuzumab and increase the inhibition effect of this drug.

Simvastatin downregulates the expression of hepcidin and erythropoietin in HepG2 cells

Statin therapy may improve responsiveness to erythropoietin-stimulating agents in patients with end-stage renal disease. Although statins increase hepatic iron uptake and storage capacity in cholestatic rats, the underlying mechanisms are unclear. Therefore, we examined the effects of a statin (simvastatin) on the expression of hepcidin, erythropoietin receptor (EPOR) and erythropoietin (EPO) in cultured HepG2 cells. HepG2 cells (6-6.5 × 10(5) cells) were seeded in 6-cm dishes and incubated overnight. The cells were then treated with 0, 0.5, 1, 3, 5, or 10 μM simvastatin, and the mRNA expression of hepcidin, EPOR, and EPO was determined. Data were collected from three independent experiments. The cDNA extracted from the cells was used as a template for real-time polymerase chain reaction, and each sample was tested in duplicate. Significant differences (P < 0.05) among groups were determined using one-way analysis of variance with Fisher's least significant difference post hoc test. Data were adjusted using Bonferroni's method. The relative mRNA expression of hepcidin in HepG2 cells treated with 0.5, 1, 3, 5, and 10 μM simvastatin, relative to the control group, was 0.7273, 0.3303, 0.2418, 0.4131, and 0.4064, respectively. The relative mRNA expression of EPOR was 0.5196, 0.2319, 0.2398, 0.4253, and 0.1245, respectively, while that of EPO was 0.9751, 0.4712, 0.4613, 0.4875, and 0.1654. There was a reverse dose-dependent effect of simvastatin. These results suggest that statins increase erythropoiesis by targeting hepcidin and iron regulatory pathways, independent of erythropoietin.

Effects of hyperoxia on erythropoietin receptor expression in lung development of neonatal rats

Oxygen toxicity is thought to be a major contributing factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Animal experiments reveal that erythropoietin (EPO) may have protective effects against hyperoxic lung injury, but the mechanisms remain unknown. The aim of this study was to evaluate effects of hyperoxia on erythropoietin receptor expression in lung development of neonatal rats. Several litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into two groups (n = 24 in each): air-exposed control group and hyperoxia-exposed group. In hyperoxia-exposed group, the rats were exposed to 85% oxygen. Pups (n = 8) from each group were sacrificed on postnatal days 3, 7, and 14. The pulmonary histological and morphometric changes were observed after hematoxylin-eosin (HE) staining under light microscope. Radical alveolar counts (RAC) were compared between the two groups to evaluate the differences of alveolarization. Expressions of platelet endothelial cell adhesion molecule-1 (PECAM-1) and erythropoietin receptor (EPOR) in lung tissue were measured by immunohistochemistry. Expressions of EPOR mRNA and EPOR protein were measured by RT-PCR and Western blotting. In hyperoxia-exposed group, there were a few inflammatory cells infiltration in interstitium on day 3 and inflammatory response worsened on day 7. Alveolar and capillary hypoplasia and interstitial fibrosis were evident on day 14. RAC increased in air-exposed control group along with the age in days. RAC decreased from day 7 in hyperoxia-exposed group compared with air-exposed control group [(6.85 ± 1.04) vs. (7.33 ± 1.0), P < 0.01], which was more evident on day 14 [(6.20 ± 1.58) vs. (9.07 ± 0.69), P < 0.001]. Expression of PECAM-1 protein increased in air-exposed control group along with the age in days. But in hyperoxia-exposed group, it decreased on day 7 and 14 [(15.14 ± 1.51) vs. (31.47 ± 2.43), (11.04 ± 1.76) vs. (41.41 ± 3.83), P < 0.001] compared with air-exposed control group. Expression of EPOR on day 3 in air-exposed control group was the strongest and weakened gradually with the increase of postnatal days. Expression of EPOR in hyperoxia-exposed group decreased on day 3 and became more evident on day 7 and day 14 compared with air-exposed control group [(1.62 ± 0.04) vs. (1.82 ± 0.06), P < 0.05; (0.48 ± 0.01) vs. (1.10 ± 0.07), (0.39 ± 0.04) vs. (0.87 ± 0.03), P < 0.001]. Expression of EPOR mRNA on day 3 in air-exposed control group was the strongest and was decreased significantly in hyperoxia-exposed group compared with air-exposed control group at all time points [(0.87 ± 0.07) vs. (1.1 ± 0.17), (0.18 ± 0.07) vs. (0.36 ± 0.08), P < 0.01;(0.14 ± 0.05) vs. (0.36 ± 0.09), P < 0.001]. EPOR may participate in the modulation of normal lung development. Depressed expression of EPOR and PECAM-1 may be involved in the pathogenesis of alveolar and capillary hypoplasia induced by hyperoxia.

Expression of erythropoietic cytokines in α-tocopherol transfer protein knockout mice with murine malaria infection

Malaria infection leads to anemia in humans which generally occurs during the chronic phase of the infection. The role that erythropoietic molecules play for anemia during malaria at low parasitemia levels is still controversial due to the lack of suitable animal models which might mimic this condition. In this regard, α-tocopherol transfer protein knockout mice, with undetectable levels of vitamin E in circulation, were possibly used as a model to investigate the role that erythropoietic molecules such as erythropoietin (EPO), erythropoietin receptor (EPOR), and macrophage migration inhibitory factor (MIF) play on the outcome of anemia during uncomplicated malaria infection at low parasitemias. The results indicate that the degree of parasitemia unlikely plays any important effect on mRNA expression of EPO and EPOR in different organs. Moreover, even though EPO and EPOR productions are impaired in the kidney and bone marrow, respectively, other organs such as the liver and spleen intend to compensate production of these cytokines to prevent anemia in the infected animals.

Recombinant Human Erythropoietin (rHuEPO) In Combination with Chemotherapy Increases Breast Cancer Metastasis In Pre-Clinical Mouse Models

AbstractAbstract 3345 Background:Erythropoietin (EPO) is a key regulator of erythropoiesis, and has been shown to stimulate growth, maintain viability, and promote differentiation of red blood cell precursors. The EPO receptor (EPO-R) is expressed by erythroid cells and by several non-hematopoietic cell types including various neoplastic cells. Erythropoiesis-stimulating agents (ESAs) are used clinically for the treatment of chemotherapy-induced anemia. The results of some recent randomized clinical trials have reported an increased incidence in adverse events and reduced survival in ESA-treated metastatic breast cancer patients receiving chemotherapy, potentially related to EPO-induced cancer progression. These results have raised concerns over ESA treatment in metastatic cancer patients. However, very little pre-clinical data is available regarding the impact of EPO on breast cancer metastasis. The goal of the current study was therefore to determine if EPO can influence the malignant behavior of breast cancer cells and/or influence the metastatic process. Methods:MDA-MB-468, MDA-MB-231, MDA-MB-435, and 4T-1 breast cancer cell lines were treated with recombinant human EPO (rHuEPO; 10 U/ml) or control media and screened for EPO-R mRNA expression levels by RT-PCR, and for EPO-R protein expression by Western blot and flow cytometry. MDA-MB-231 (231) and MDA-MB-435 (435) cell lines were used for functional assays in vitro and in vivo. Untreated or rHuEPO treated cells were grown in 2D and 3D in vitro systems (standard tissue culture plates and 0.6% soft agar, respectively) to determine if rHuEPO influenced growth. In vitro cell survival was also assessed in response to treatment with rHuEPO in the presence or absence of paclitaxel chemotherapy (10mg/ml), radiation (10G), or hypoxic conditions (1% O2). Following mammary fat pad injection, in vivo effects of rHuEPO (300U/kg) alone or in combination with paclitaxel treatment (10mg/kg) were assessed in mouse models of tumorigenicity and spontaneous metastasis. Results:Expression analysis of EPO-R mRNA and protein revealed a large variation in levels across different cell lines. The majority of cell lines did not express cell surface EPO-R by flow cytometry, although two cell lines (231 and 435) did show weak expression of EPO-R mRNA, with only the 231 cell line showing EPO-R expression by Western blot. In vitro, a small protective effect from rHuEPO on radiation-treated 435 cells was seen (p<0.05); however, rHuEPO treatment alone or combined with chemotherapy or hypoxia did not cause a significant increase in cell survival relative to untreated controls cells. In contrast, in vivo studies demonstrated that rHuEPO increased the incidence and burden of lung metastases in immunocompromised mice injected with 231 or 435 cells and treated with paclitaxel relative to mice treated with paclitaxel alone (p<0.05). Conclusions:The lack of an in vitro effect of rHuEPO highlights the importance of in vivo studies to delineate the effects of EPO on the metastatic process. Our novel findings demonstrate that rHuEPO can reduce the efficacy of chemotherapy in the metastatic setting in vivo, and in some cases enhance the inherent metastatic growth potential of human breast cancer cells.This work was supported by funding from the London Regional Cancer Program and Janssen Ortho Canada Disclosures:Xenocostas:Janssen Ortho: Consultancy, Honoraria, Research Funding. Allan:Janssen Ortho: Research Funding.

Simulated microgravity induce apoptosis and down-regulation of erythropoietin receptor of UT-7/EPO cells

Hematopoietic progenitor cell proliferation can be alternated on either spaceflight or under simulated microgravity experiments on the ground; however, the underlying mechanism remains largely unknown. In the present study, we have demonstrated that exposure of human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO cells to conditions of simulated microgravity with a rotary culture instrument significantly inhibited the cellular proliferation rate. Adding higher concentrations of EPO to the culture medium failed to improve the inhibitory status. Cell apoptosis was detected by fluorescence staining of cell nuclei and a flow cytometry assay using Annexin V/PI double staining. This microgravity-induced apoptosis in UT-7/EPO cells could be blocked by a pancaspase inhibitor Z-VAD-FMK. Immunoblotting demonstrated that rotary culture resulted in a reduction of the expression of Bcl-xL, an anti-apoptotic protein, and the cleavage of caspase-3. Furthermore, rotary culture reduced surface localization and protein content, as well as the mRNA expression of erythropoietin receptor (EPOR) of UT-7/EPO. Take together, the findings indicated that simulated microgravity may induce mitochondrial related apoptosis of UT-7/EPO cell through depressing the EPO–EPOR pathway.

Activation of the erythropoietin receptor in human skeletal muscle

Erythropoietin receptor (EPOR) expression in non-hematological tissues has been shown to be activated by locally produced and/or systemically delivered EPO. Improved oxygen homeostasis, a well-established consequence of EPOR activation, is very important for human skeletal muscle performance. In the present study we investigate whether human skeletal muscle fibers and satellite cells express EPOR and if it is activated by exercise. Ten healthy males performed 65 min of cycle exercise. Biopsies were obtained from the vastus lateralis muscle and femoral arterio-venous differences in EPO concentrations were estimated. The EPOR protein was localized in areas corresponding to the sarcolemma and capillaries. Laser dissection identified EPOR mRNA expression in muscle fibers. Also, EPOR mRNA and protein were both detected in human skeletal muscle satellite cells. In the initial part of the exercise bout there was a release of EPO from the exercising leg to the circulation, possibly corresponding to an increased bioavailability of EPO. After exercise, EPOR mRNA and EPOR-associated JAK2 phosphorylation were increased. Interaction with JAK2 is required for EPOR signaling and the increase found in phosphorylation is therefore closely linked to the activation of EPOR. The receptor activation by acute exercise suggests that signaling through EPOR is involved in exercise-induced skeletal muscle adaptation, thus extending the biological role of EPO into the skeletal muscle.

Involvement of Erythropoietin in Retinal Ischemic Preconditioning

The purpose of this study was to examine the role of erythropoietin in retinal ischemic preconditioning (IPC). Rats were subjected to retinal ischemia after IPC. Electroretinography assessed functional recovery after ischemia; retinal sections were examined to determine loss of retinal ganglion cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to assess apoptosis. Levels of downstream mediators were measured in retinal homogenates by Western blotting. To assess the involvement of erythropoietin in IPC, Western blotting was used to measure levels of erythropoietin and its receptor (EPO-R) in retinal homogenates after IPC. To examine erythropoietin's role in IPC, the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC was studied. Erythropoietin levels did not change after IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histologic neuroprotection produced by IPC in comparison to control injection of denatured sEPO-R. Apoptotic damage after ischemia was enhanced in the sEPO-R-treated retinas as indicated by fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Phosphorylated extracellular-signal-regulated kinase and heat shock protein 27, but not protein kinase B, upregulated in denatured sEPO-R-treated retinae, were attenuated in eyes injected with sEPO-R. These results indicate that EPO-R upregulation is a critical component of the functional, histologic, and antiapoptotic protective effect of IPC on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.

Erythropoetin receptor expression in the human diabetic retina

BackgroundRecent evidence suggests erythropoietin (EPO) and the erythropoietin receptor (EPOR) may play a direct role in the pathogenesis of diabetic retinopathy. Better characterization of the EPO-EPOR signaling system in the ischemic retina may offer a new therapeutic modality for ischemic ophthalmic diseases. This study was performed to identify EPOR mRNA expression in the human diabetic eye.FindingsEPOR antisense RNA probes were validated on human pancreas tissue. In the normal eye, EPOR was expressed in the retinal ganglion cell layer. Minimal expression was observed in the inner and outer nuclear layer. Under conditions of diabetic retinopathy, EPOR expression shifted to photoreceptor cells. Increased expression was also observed in the peripheral retina.ConclusionEPOR expression may be a biomarker or contribute to disease mechanisms in diabetic retinopathy.

A novel heterozygous HIF2AM535I mutation reinforces the role of oxygen sensing pathway disturbances in the pathogenesis of familial erythrocytosis

HIF2A transcription factor plays a central role in the regulation of the hypoxia responding pathway in mammalian cells, by modulating erythropoiesis and angiogenesis. Molecular alterations of oxygen sensing pathway constituents are implicated in hereditary erythrocytosis. Here we show that 2 members of a family with idiopathic erythrocytosis exhibited a new heterozygous G to A mutation at base 1605 of the exon 12 of hypoxia-inducible factor-2A (HIF2A) gene. This mutation determines the replacement of methionine by isoleucine at the position 535, very close to the position 531, where the hydroxyl acceptor prolyne is located. In addition, we found that mRNA expression of erythropoietin receptor, vascular endothelial growth factor, transferrin receptor, adrenomedullin and N-myc downstream regulated gene 1, up-regulated by HIF2A or hypoxia, were significantly higher in patients carrying the mutation than in normal controls. These results suggest that the HIF2A(M535I) gene mutation could induce hereditary erythrocytosis at a young age.