Expression Levels Of Erythropoietin Research Articles

AKT/GSK-3β/β-catenin signaling pathway participates in erythropoietin-promoted glioma proliferation.

Although erythropoietin (EPO) has been proven to significantly promote the proliferation of cancer cells, the mechanism for promoting glioma proliferation is poorly understood. Here, we examined the functional role of the AKT/GSK-3β/β-catenin signaling pathway in the EPO-mediated proliferation of glioma. The distribution of EPO and Ki-67 among clinical samples with different WHO grades was plotted by Immunological Histological Chemistry analysis. U87 and U251 glioma cell lines were treated with short hairpin RNA targeting (shEPO), recombinant human erythropoietin (rhEPO) and/or AKT-specific inhibitor (MK-2206). The changes in phosphorylated AKT, nuclear β-catenin, cyclin D1 and p27kip1 expression were detected.Cell cycle distributions and glioma proliferation in vitro and in vivo were analyzed. The expression level of EPO was significantly elevated with the increase of WHO grade and Ki67 in clinical glioma specimens. In vitro, knockdown of endogenous EPO in U87 and U251 cells effectively block the phosphorylation of AKT and GSK-3β and the expression of nuclear β-catenin. shEPO treatment also significantly decreased the expression of cyclin D1 and increased the expression of p27kip1. The cell cycle transition then slowed down and the proliferation of glioma cells or mouse xenograft tumors both decreased. Treatment of cells or tumors with extra rhEPO reversed the above biological effects mediated by shEPO. rhEPO-induced activation of the AKT/GSK-3β/β-catenin pathway and proliferation were abolished by MK-2206. Our study identified the AKT/GSK-3β/β-catenin axis as a critical mediator of EPO-induced glioma proliferation and further provided a clinically significant dimension to the biology of EPO.

Plasma Erythropoietin, IL-17A, and IFN\u03b3 as Potential Biomarkers of Motor Function Recovery in a Canine Model of Spinal Cord Injury

Traumatic spinal cord injury (SCI) is a devastating neurological disease for which an accurate, cost-effective prediction of motor function recovery is in pressing need. A plethora of neurochemical changes involved in the pathophysiological process of SCI may serve as a new source of biomarkers for patient outcomes. Five dogs were included in this study. We characterized the plasma cytokine profiles in acute phase (0, 1, and 3 days after SCI) and subacute phase (7, 14, and 21 days after SCI) with microarray analysis. The motor function recovery following SCI was monitored by Olby scores. The expression level of differentially expressed proteins (DEPs) was measured with enzyme-linked immunosorbent assay (ELISA). Then, correlations with the Olby scores and receiver operating characteristic curve (ROC) analysis were performed. We identified 12 DEPs including 10 pro-inflammatory and 2 anti-inflammatory cytokines during the 21-day study period. Among those, the expression levels of erythropoietin (EPO), IL-17A, and IFNγ significantly correlated with the Olby scores with R2 values of 0.870, 0.740, and 0.616, respectively. The results of the ROC analysis suggested that plasma EPO, IL-17A, and IFNγ exhibited a significant predictive power with an area under the curve (AUC) of 0.656, 0.848, and 0.800 for EPO, IL-17A, and IFNγ, respectively. Our results provide a longitudinal description of the changes in plasma cytokine expression in the acute and subacute stages of canine SCI. These data reveal novel panels of inflammation-related cytokines which have the potential to be evaluated as biomarkers for predicting motor function prognosis after SCI.

Unraveling Intron Retention: Differential Expression Pattern in Brain and Kidney Cells After Hypoxia or Ischemia

IntroductionThe inclusion of introns in the mature mRNA (intron retention), has been recently uncovered as a mechanism for the regulation of genetic expression in vertebrates. Intron retention is implicated in tissue specific protein diversity, splicing regulation and control of gene expression. We have recently identified intron retention as a mechanism involved in the regulation of erythropoietin (EPO). EPO is the key regulator of erythropoiesis and is transcriptionally regulated by hypoxia inducible factor 2 (HIF2). The ability to easily detect intron retention could add to our ability to better understand genetic regulation of our genomes in stress conditions, such as ischemic diseases (ex/ myocardial infarction (MI) or stroke).ObjectiveTo evaluate the role of intron retention mechanism in EPO expression level to gain a better understanding of its regulation in response to hypoxia and ischemia, conditions which result in EPO expression.MethodsHTB16 (neuroblastoma), CHP212 (glioblastoma), hCMEC/D3 (blood brain barrier) and HEK293 (kidney) were placed in hypoxic (1% oxygen), ischemic (1% oxygen, no serum or glucose) or normoxic conditions (20% oxygen) for 24 hours prior to RNA extraction. Quantitative polymerase chain reaction (qPCR) was performed with two sets of primers for EPO, the first set was targeting intron inclusion with each primer in a separate exon (intron primers) and the second set was targeting a region that spans two exons, which would not amplify if introns were present (non‐introns). Relative quantification for the level of expression was determined by qPCR using β‐actin and YWHAZ as reference genes for normalization purposes. Statistical analysis of transcript expression and the ratio between intron and non‐intron detecting primer sets were determined using analysis of variance (ANOVA).ResultsEPO mRNA level was increased in HTB16, CHP212 and HEK293, but not in hCMEC/D3 cell lines in response to ischemia, but not hypoxia. However, when the ration of intron vs. non‐intron expression level was determined a trend towards more intron retaining transcripts in HTB16, CHP212 and hCMEC/D3 but not in HEK293 was observed, suggesting an induction for the expression of this gene under this ischemic condition.ConclusionsIntron retention mechanism in EPO is taking place in brain‐derived cell lines, but not in kidney‐derived cells. These results indicate a tissue specificity of intron retention mechanism that could regulate differential expression level of EPO under different physiological and stressful conditions. These findings highlight the complexity of EPO regulation. Hence, intron retention must be considered when examining the expression of a gene to better understand expression in ischemic disease models, such as MI or stroke.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)

EPO and EPOR Expression in Patients with Acute Leukemia and Its Clinical Significance

To investigate the expression of erythropoietin (EPO) and erythropoietin receptor (EPOR) in patients with acute leukemia (AL) and its clinical significance. The levels of EPO and EPOR in plasma were determined by ELISA kit. mRNA expression levels of EPO and EPOR were determined by RT-RCR. The protein expression levels of EPO and EPOR were detected by Western blot. The EPO protein levels in marrow plasma of ALL and AML group were significantly higher than those in the control group (P＜0.05), EPOR protein levels in ALL and AML group were significantly lower than those in the control group (P＜0.05). The mRNA levels of EPO and EPOR in ALL and AML groups were significantly higher than those in the control group (P＜0.05). The mRNA levels of EPO and EPOR in the high risk ALL and AML groups were significantly higher than those in the medium, low risk group and the control group (P＜0.05). The protein expression levels of EPO and EPOR in ALL and AML groups were significantly higher than that in control group (P＜0.05). The mRNA levels of EPO and EPOR in ALL and AML groups did not correlate with hemoglobin level and erythrocyte count (P＞0.05). The expressions of EPO and EPOR is higher in ALL and AML patients. The expression levels of EPO and EPOR relate with the risk of ALL and AML. High risk patients have higher expression levels of EPO and EPOR, however, the expression levels of EPO and EPOR do not correlate with hemoglobin level and erythrocyte counting.

Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells

AimsThe aim of this study was to evaluate gamma-aminobutyric acid (GABA)- and piperine-induced erythropoietin (EPO) and EPO-receptor expression. Materials and methodsThe effect of GABA and piperine on cell viability was examined using kidney epithelial cells. Expression levels of EPO and EPO-R mRNA and protein were evaluated in response to GABA and piperine treatments. GABA- and piperine-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathway was investigated. Additionally, EPO function was evaluated using conditioned media containing EPO. The GABA receptor type involved in this process was identified. Key findingsMessenger RNA and protein expression levels of EPO and EPO-R significantly increased in response to treatment with GABA, piperine, or the combination of both, compared with control. GABA plus piperine synergistically enhanced EPO and EPO-R expression through p38 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, but not through the extracellular signal-regulated kinase (ERK) MAPK pathway. SB203580 and SP600125 (p38 and JNK pathway inhibitors, respectively) attenuated GABA plus piperine-induced EPO and EPO-R expression. Treatment of macrophages with EPO-containing conditioned media induced mRNA expression of interleukin (IL)-10 and nuclear factor (NF)-κB due to the interaction between EPO and EPO-R. Interestingly, GABA-induced EPO and EPO-R expression was mediated through GABAA, not GABAB, receptor activation. SignificanceThese findings demonstrate that GABA plus piperine-mediated p38 and JNK MAPK activation increases EPO and EPO-R expression, resulting in up-regulation of IL-10 and NF-κB.

Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells

Chinese hamster ovary (CHO) cells are mainly used for recombinant protein production. However, the unstable transgene expression and lower transgene copy numbers are the major issues need to be resolved. Here, eleven internal ribosome entry site (IRES) elements from viral and cellular IRES were evaluated for foreign gene expression in CHO-S cells. We constructed eleven fusing plasmids containing different IRES sequences downstream of the enhanced green fluorescent protein (EGFP) gene. EGFP expression was detected by flow cytometry and the transgene copy number was evaluated by quantitative PCR. The erythropoietin (EPO) protein was also used to assess the stronger IRES. The results showed that IRES from human rhinovirus (HRV) exhibited the highest EGFP expression level under transient and stable transfections. The EGFP expression level of vector with IRES from HRV was related to the gene copy number in stably transfected CHO-S cells. Moreover, IRES from HRV induced higher expression level of EPO compared with one mutant IRES from EMCV in transfected cells. In conclusion, IRES from HRV can function as a strong IRES element for stable expression in CHO-S cells, which could potentially guide more effective foreign gene expression in CHO-S cells.

Analysis of Glycosylation Profiles of Serum Glycoprotein from Iron Overload Thalassemia.

The aim of this study was to use a lectin microarray to detect differential glycan profiling of serous glycoprotein in iron overload thalassemia patients. This study enrolled iron overload α/β-thalassemia patients and no iron overload α/β-thalassemia patients. Lectin microarray was used to detect the alteration of protein glycosylation. The reliability of the lectin microarray results was verified by the lectin blotting technique. Expression level of hepcidin, erythropoietin, ferritin, and transferrin were measured by western blotting. Data were analyzed using the SPSS 16.0 software. In this study, 19 differentiating lectins were screened from the iron overload α-thalassemia group, and 15 were screened from the iron overload β-thalassemia group. The agglutinin blotting technique demonstrated that the results of the Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), and Wheat germ agglutinin (WGA) agglutinin affinity for serum glycoproteins were consistent with the results of the lectin microarray. In iron overload thalassemia groups, expression levels of erythropoietin and ferritin were increased, but hepcidin and transferrin were significantly reduced. The differentially expressed glycoprotein database of iron overload thalassemia was successfully created, and the specific glycan patterns of serous glycoprotein might be efficient biomarkers for diagnosis or progression of iron overload thalassemia.

Hypoxia pretreatment and EPO-modification enhance the protective effects of MSC on neuron-like PC12 cells in a similar way

Mesenchymal stem cells (MSC) based cell transplantation therapy is proved to be an attractive strategy with great potential for improvement of hypoxia induced neural damage. In the present study, MSCs were co-culture with PC12 to investigate its protective effects against hypoxia pretreatment, and the Lactate dehydrogenase (LDH) release assay, MTT and Anexin V staining were performed to analysis the cellular damage or apoptotic. RT-PCR and Western blotting were further used to investigate the underlying mechanism. The results indicate that hypoxia treatment results in the decrease of PC12 cell viability, yet co-culture with MSC could protect the PC12 from hypoxia induced damage. Hypoxia pre-activated or EPO transduced MSC with up-regulated erythropoietin (EPO) expression could further enhance MSC's protective effect against hypoxia induced cell damage, which was associated with high level of anti-apoptotic p-Akt and ration Bcl-2/Bax, and decreased Caspase 3 in PC12. Taken together, these data suggests high levels of MSC-mediated cyto-protection is closely tied to high gene expression levels of EPO. The up-regulation of EPO for enhanced MSC-mediated cyto-protection may has great potential for the MSC cellular therapy of neural or neuronal injuries induced by hypoxia.

A Novel EPO Gene Mutation In a Family With Autosomal Dominant Polycythemia

Familial polycythemia/erythrocytosis (ECYT by OMIM), characterized by an absolute increase in red cell mass, is a heterogeneous group of disorders that can be attributed to either intrinsic erythroid progenitor defects, resulting in their hyperproliferation (primary polycythemias), or from circulating extracellular factors such as erythropoietin (EPO) stimulating erythropoiesis (secondary polycythemias). Known primary familial causes include gain-of-function EPO receptor gene mutations. Secondary polycythemia causes include gene mutations that increase hemoglobin oxygen affinity (a, b, and g globins and 2, 3 bisphosphoglycerate mutase, and cytochrome b5 reductase). Several mutations result in alterations of the hypoxia-sensing pathway, which is a primary regulator of EPO production, and familial polycythemia. These include gain-of-function mutations of the HIF-2-a gene (HIF2A), and loss-of-function mutations of two negative regulators of HIFs (EGLN1/PHD2) and von Hippel-Lindau (VHL) genes. Some of these mutations exhibit overlapping features of both primary and secondary polycythemias, such as Chuvash polycythemia. No mutations of the EPO gene have yet been reported.Here we report a 5-generation Caucasian family with autosomal dominant polycythemia. The propositus (III-11) was initially seen 28 years ago at the age of 2 years old. He had moderately increased EPO levels, no splenomegaly, and normal leukocyte and platelet numbers. He was not hypoxic and his hemoglobin oxygen dissociation (P50) was normal. His 21 family members agreed to be tested and 10 were polycythemic, while 11 were normal. Later linkage analyses of 18 re-consented relatives examined the association of a polycythemic phenotype with polymorphisms of HIF2A, HIF1A, EPOR, PHD2, and VHL genes, ruling these out as the cause. Exome sequencing of 5 affected individuals revealed a novel nucleotide change in chromosome 7 at -136nt upstream of the EPO gene (NG_021471 -136 G>A) from the ATG initiation site. This variant has not been reported in any publicly available genome databases and none of the 8 unrelated Caucasian controls have this variant. To determine the distribution of this variant and its segregation, we screened 7 affected and 8 unaffected relatives; all 8 unaffected samples were negative for this EPO variant, while all 7 affected individuals were heterozygous for this variant in the 5'UTR of the EPO gene.The effect(s) of this 5'UTR variant on EPO gene transcription is being examined by Luciferase assay, as well as in an expression assay using HEK293 EPO producing cells. In brief, constructs containing the -136nt variant having either the G (wild-type) or A (mutant) nucleotide of the EPO gene have been made and inserted into the pGL3 vector for the luciferase assay. EPO transcript quantitation studies will be carried out using human EPO cDNA clones with or without the -136nt variant transfected into HEK293 cell line and analyzed under two different conditions, ambient and at 5% oxygen. EPO expression levels under these two different conditions will be determined at multiple time points.This is the first report of an EPO gene mutation associated with familial polycythemia; its functional impact is being studied. Disclosures:No relevant conflicts of interest to declare.

Serum erythropoietin level predicts the prognosis of chronic heart failure with or without anemia

The aim of this study was to explore the correlation of erythropoietin (EPO) with N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high sensitivity C-reactive protein (hs-CRP) in patients with chronic heart failure (CHF) or CHF complicated with anemia, in addition to its correlation with the prognosis of the patient. A total of 217 CHF patients were enrolled in this study. The patients were graded according to the cardiac function criteria of the New York Heart Association (NYHA). The serum EPO, NT-proBNP and hs-CRP levels of the patients were determined. The patients were followed up for ≥24 months. The EPO expression level in patients with NYHA II–IV CHF was significantly higher compared with that in the control group (P<0.05). EPO expression increased with the aggravation of CHF, exhibiting significant differences amongst the various NYHA graded groups (P<0.05). The EPO expression level increased significantly with an increase in NHA grade in addition to the severity of the anemia in the patients with CHF complicated by anemia (P<0.05). In the patients who succumbed (mortality group), the expression level of EPO was significantly higher and the hemoglobin level was significantly lower compared with those of the survival group (P<0.05). The EPO expression levels were elevated in CHF patients and patients with CHF and anemia. The level of expression correlated positively with the severity of CHF as well as that of anemia. Serum EPO measurements were successful in predicting the mortality and re-hospitalization rates of CHF patients at the end point, within two years of follow-up.

Erythropoietin is involved in hemoprotein syntheses in developing human decidua

Before establishment of feto-placental circulation, decidua can synthesize hemoproteins to maintain oxygen homeostasis in situ. Using the human decidua of induced abortions ranging from 5 to 8 weeks of gestation, we determined the expression levels of erythropoietin, erythropoietin receptor, cytoglobin, myoglobin, embryonic-, fetal- and adult hemoglobin mRNA by quantitative RT-PCR analysis and identified their proteins by Western blot and immunohistochemical analyses. Erythropoietin signaling was demonstrated in phosphatidylinositol-3-kinase/protein kinase B pathway by Western blot, and the transcriptional factors for erythroid and non-erythroid heme synthesis were examined by RT-PCR analysis. In decidua, erythropoietin and its receptor mRNAs, erythropoietin receptor protein and phosphatidylinositol-3-kinase, were expressed with a peak at 6 weeks of gestation. Moreover, the decidua during 5 to 8 weeks of gestation expressed embryonic, fetal and adult hemoglobins additionally cytoglobin and myoglobin at transcriptional and protein levels. The heme portion of these hemoproteins is considered to be synthesized by non-erythroid δ-aminolevulinate synthase. These hemoproteins were discernible especially in decidual cells concomitant with cytotrophoblast cells and macrophage in these developing decidua. Considering the different capacity for oxygen binding and dissociation among hemoglobins with the oxygen storage capacity for cytoglobin and myoglobin, these hemoproteins appear to play a role in oxygen demand in decidua in situ before development of feto-placental circulation under the control of erythropoietin signaling.

A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells

The use of non-integrating human artificial chromosomes (HACs) in gene therapy possibly allows for safe and reliable genetic modification of human cells without insertional mutagenesis and/or unexpected oncogene activations. Although we previously demonstrated that the HAC provides long-term therapeutic erythropoietin (EPO) production in normal human primary fibroblasts (hPFs), the expression level of EPO was too low to provide medical benefits for human therapy. Thus, the next challenge for the application of this system in therapeutic purposes is to improve the transgene expression on HACs. Here, we newly constructed chromosome 14-based HACs and examined the effects of the telomere and promoter regions on the expression level of the tansgene in hPFs. We showed that the use of natural telomere/sub-telomere and enhancers within the 5′ untranslated region of the human ubiquitin C gene greatly increased (over 1000-fold) the EPO production in hPFs. Furthermore, we demonstrated the reprogramming of mouse embryonic fibroblasts by HAC-mediated introduction of four transcription factors, and established induced pluripotent stem cells with no trace of the HACs carrying multiple expression cassettes with large genome fragments. These results indicate that this HAC system could allow us to manipulate multiple transgenes efficiently in human primary cells, providing a promising tool not only for gene therapy but also for investigating genome functions in drug discoveries.

The relationship of erythropoietin overexpression with von Hippel-Lindau tumour suppressor gene mutations between hypoxia-inducible factor-1α and -2α in sporadic clear cell renal carcinoma

Decreased levels of von Hippel-Lindau (VHL) tumour suppressor protein are associated with up-regulation of hypoxia-inducible factor (HIF), leading to increased tumour proliferation, angiogenesis and progression. The role of erythropoietin (EPO), a target gene for HIF, remains unknown for sporadic clear cell renal cell carcinoma (sCCRCC). In this study, we determined expression levels of EPO, and its correlation with VHL mutations and HIF-1α and HIF-2α expression in 82 patients identified with sCCRCC following nephrectomy. We identified VHL gene alterations using multiplex polymerase chain reaction, purifying products of polymerase chain reaction, and direct sequencing. Immunohistochemical staining for HIF-1α, HIF-2α and EPO was performed for tumour and corresponding normal tissues. Data were analyzed with respect to clinicopathological factors. EPO was detected in 87.8% of sCCRCC tumours versus 7.3% for normal tissues. EPO expression was related to tumours demonstrating VHL gene abnormalities. Of specimens with VHL alterations 95.6% tested positive for EPO, versus 78.3% when VHL gene expression was normal (P<0.01). EPO was identified in 96.2 and 94.2% of HIF-1α and HIF-2α positive specimens, respectively, compared to 72.4 and 53.8% for HIF-1α and HIF-2α negative groups (p<0.01). Moreover, EPO expression correlated significantly with increasing nuclear grade (p<0.05). HIF-2α was identified in 84.1% of sCCRCC, compared to 64.6% for HIF-1α. Expression of HIF-1α, HIF-2α and EPO is common in sCCRCC. Although both forms of HIF up-regulate expression of EPO, the relationship to HIF-2α appears to be more pronounced. The VHL-HIF-EPO pathway requires further study, as it may represent a potential molecular target for therapy of sCCRCC.

Effects of flavones extracted from Portulaca oleracea on ability of hypoxia tolerance in mice and its mechanism

To identify anti-hypoxia ingredients extracted from Portulaca oleracea and to find out the possible mechanism of its anti-hypoxia actions. Seventy mice were randomly divided into seven groups which were untreated (normal saline), ginsenosides-treated, polysaccharide-treated, acidic components-treated, basic components-treated, alkaloids-treated and flavones-treated groups, and the ingredients of polysaccharide, acidic components, basic components, alkaloids and flavones were extracted from Portulaca olerace. The mice in each group were fed with corresponding ingredients for one week respectively. Then the survival time of mice in hypoxic conditions was observed. Another 90 mice were divided into 3 groups: untreated (normal saline), ginsenosides-treated and flavones-treated groups. The mice in each of these 3 groups were divided into 3 subgroups according to 12-, 24- and 36-hour exposure to hypoxia (10% oxygen and 90% nitrogen), respectively. After exposure to hypoxia, the red blood cell count (RBC), hemoglobin (Hb) concentration and hematocrit (HCT) in mice were determined. The plasma erythropoietin (EPO) levels of mice were detected by enzyme-linked immunosorbent assay (ELISA) and the relative values of EPO mRNA in renal tissue and pallium of mice were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The survival time of mice in hypoxic conditions in flavones-treated group was significantly longer than that in the untreated group. The RBC, Hb concentration, HCT, plasma EPO level and the relative values of EPO mRNA in renal tissue and pallium of mice were significantly higher in the flavones-treated group than those in the untreated group. The anti-hypoxia ingredients extracted from Portulaca oleracea are flavones and the anti-hypoxia effects may be obtained by improving the expression level of EPO and accelerating the generations of erythrocyte and Hb.

Effect of low adapted temperature and medium composition on growth and erythropoietin (EPO) production by Chinese hamster ovary cells

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC). When supplements were added to the medium at 33 degrees C, the growth of IBE and CO5 cells increased by 27% and 26%, respectively and the maximum yield of EPO was increased by 40% in both cell lines. The absolute EPO concentration in the CO5 cells was always 55-60% higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at 33 degrees C until their growth rates approached those at 37 degrees C, the growth rates of both IBE and CO5 cells increased by 54% and their maximum EPO levels increased by up to 73% and 56%, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.