EpoR mRNA Research Articles

Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

Modulation of alloimmune responses is critical to improving transplant outcome and promoting long-term graft survival. To determine mechanisms by which a nonhematopoietic erythropoietin (EPO) derivative, carbamylated EPO (CEPO), regulates innate and adaptive immune cells and affects renal allograft survival, we utilized a rat model of fully MHC-mismatched kidney transplantation. CEPO administration markedly extended the survival time of kidney allografts compared with the transplant alone control group. This therapeutic effect was inhibited when the recipients were given LY294002, a selective inhibitor of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway or anti-EPO receptor (EPOR) antibody, in addition to CEPO. In vitro, CEPO inhibited the differentiation and function of dendritic cells and modulated their production of pro-inflammatory and anti-inflammatory cytokines, along with activating the PI3K/AKT signaling pathway and increasing EPOR mRNA and protein expression by these innate immune cells. Moreover, after CD4+ T cells were exposed to CEPO the Th1/Th2 ratio decreased and the regulatory T cell (Treg)/Th17 ratio increased. These effects were abolished by LY294002 or anti-EPOR antibody, suggesting that CEPO regulates immune responses and promotes kidney allograft survival by activating the PI3K/AKT signaling pathway in an EPOR-dependent manner. The immunomodulatory and specific signaling pathway effects of CEPO identified in this study suggest a potential therapeutic approach to promoting kidney transplant survival.

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.

11P - Expression of erythropoietin receptor variant forms is associated with lymphatic invasion and metastasis formation in breast cancer

Background: Anemia not only impairs the quality of life of patients, but it leads to tumor hypoxia, resistance to therapy and reduces survival. Although recombinant Epo has revolutionized the treatment of anemia, clinical trials suggested a potential adverse effect of Epo on tumor recurrence and patient survival. Previously we identified altered EpoR mRNA transcripts in breast cancer cells lacking ligand binding domain exon 1-3 sequences in vitro. Our hypothesis is that altered expression of these proteins may be associated with unique clinicopathologic features. Methods: The expression level of C-and N-terminal portion of EpoR were measured by quantitative RT-PCR in 157 retrospective cohort of archived breast cancers. In order to identify the variant EpoR mRNA forms we have established a SMART RACE PCR assay using EpoR specific primer sets producing large overlapping sequences. Total RNA was frozen sections of 20 primary breast cancers after the H&E diagnoses.The products of the SMART RACE and nested PCR reactions was subjected to sequence analysis. Results: Out of the 157 samples 95 (61%) showed more than 2-fold change between the expression levels of the C and N-terminal regions of EpoR by quantitative RT-PCR. Significant correlation was found between the expression levels of EpoR mRNA variants and lymphatic invasion of the tumors. Logistic regression model demonstrated the association of metastatic capacity and expression differences of extra and intracellular regions of EpoR in primary breast cancers (P < 0.05, greater than 2-fold change). Using a SMART RACE PCR assay a single EpoR mRNA species at ∼1500 kb, corresponding to the full length wild type EpoR mRNA, and several distinct bands at ∼1300 kb, ∼1000 kb and ∼800 kb, lacking exon 1-4 N-terminal ligand binding domain, were seen in the samples. Conclusions: We showed that different variant forms of EPOR present not only in vitro but also in the individual breast tumor samples. These altered forms of EpoR may be responsible for the observed differences in the Epo responsiveness of EpoR bearing breast cancers. Legal entity responsible for the study: Zsuzsa Rakosy. Funding: OTKA PD 116996, 2015-2018. Disclosure: The author has declared no conflicts of interest.

Correction of Anemia in Chronic Kidney Disease With Angelica sinensis Polysaccharide via Restoring EPO Production and Improving Iron Availability.

Given the limited efficacy and potential disadvantages of erythropoiesis-stimulating agents (ESAs) in treating anemia of chronic kidney disease (CKD), the development of better alternative therapies has become a priority. The primary purpose of this study is to investigate the effects of Angelica sinensis polysaccharide (ASP) and its underlying mechanism in the treatment of renal anemia. In the present study, we found that ASP could enhance hypoxic induction of EPO in Hep3B cells, with a mechanism that involved the stabilization of HIF-2α protein. In parallel, ASP rescued the inhibition of EPO, induced by proinflammatory factor TNF-α through blocking GATA2 and NF-κB activation. In a rat model of adenine-induced anemia of CKD, oral administration of ASP corrected anemia and alleviated renal damage and inflammation. By increasing the accumulation of HIF-2α protein and reducing the expression of NF-κB and GATA2 as well as pro-inflammatory cytokines, ASP stimulated both renal and hepatic EPO production, and resulted in an elevation of serum EPO. The restoration of EPO production and EPOR mRNA expression with ASP treatment activated EPOR downstream JAK2/STAT5 and PI3K/Akt signaling, induced their target genes, such as Bcl-xL, Fam132b and Tfrc, and increased Bcl-2/Bax ratio in bone marrow-derived mononuclear cells of CKD rats. Furthermore, we found that ASP suppressed hepatic hepcidin expression, mobilized iron from spleen and liver and increased serum iron. These findings demonstrate that ASP elicits anti-anemic action by restoring EPO production and improving iron availability in the setting of CKD in rats.

The Effects of Curcumae Longae Radix, Curcuma phaeocaulis Radix and Their Processed Products on Epo/EpoR Pathway and CD62p.

Background: Curcumae Radix (Yujin) has been widely used to treat Qi stagnation due to Liver depression (LDQS) in traditional Chinese medicine (TCM) for a long time and is good at dispelling melancholy by soothing liver to regulate qi and relieving pain by promoting blood circulation for removing blood stasis. Yujin stir-frying with vinegar can strengthen the effect of dispersing stagnated hepatoqi to stop pain by injecting medicine into the liver and stir-frying with wine can strengthen the effect of promoting blood circulation for removing blood stasis. Because the reason for the enhancement of clinical efficacy by processing is unclear, it is difficult to select and use processed products in the clinic.Aim/Hypothesis: In this study, from the point of view of the platelet function, we start to investigate the mechanism for promoting blood circulation, removing blood stasis, and enhancing clinical efficacy by processing.Methods: This study mainly takes Curcumae Longae Radix (HSYJ) and Curcuma phaeocaulis Radix (LSYJ) as the research subjects. They are genuine drugs in Sichuan Province, China. A high-performance liquid chromatography (HPLC) is used to analyze the main chemical constituents of Yujin and its processed products, and show the difference between the two species, with and without processing. A rat model of blood stasis induced by LDQS is established by giving the tail clamp stimulation, injecting epinephrine hydrochloride subcutaneously, and treating with an effective dose (0.9 g⋅kg-1) according to the conversion of human clinical dosage for 2 weeks. After the experiment, the serum levels of erythropoietin are measured by ELISA. Furthermore, RT-PCR and WB are used to detect EpoR mRNA and protein expression in the hepatic tissue. Flow cytometry is used to measure peripheral blood CD62p expression.Results: There is a great difference between the chemical compositions of the two species, the number of chromatographic peaks of Curcumae Longae Radix is more than that of Curcuma phaeocaulis Radix. Curcuminoids is the main component of HSYJ, while curcuminoids is almost free from LSYJ. Curcuminoids is almost insoluble in water. After stir-frying with vinegar or wine, it can increase the dissolution of curcuminoids in water. In rat models, the levels of Epo, EpoR, and CD62p are significantly increased. After intragastric administration of Yujin, indicators show varying degrees of callback. HSYJ is better than LSYJ, and the processed product by stir-frying with wine is better than other processed products.Conclusion: The results show that the mechanism of promoting blood circulation for removing blood stasis in Yujin may be able to inhibit the activation and aggregation of platelets by intervening the Epo/EpoR pathway and regulating CD62p down. Stir-frying with wine can enhance this effect.

No erythropoietin-induced growth is observed in non-small cell lung cancer cells.

Lung cancer patients have the highest incidence of anemia among patients with solid tumors. The use of recombinant human erythropoietin (Epo) has consistently been shown to reduce the need for blood transfusions and to increase hemoglobin levels in lung cancer patients with chemotherapy-induced anemia. However, clinical and preclinical studies have prompted concerns that Epo and the presence of its receptor, EpoR, in tumor cells may be responsible for adverse effects and, eventually, death. The question has been raised whether Epo promotes tumor growth and inhibits the death of cancer cells. In this study, we investigated the presence and functionality of EpoR, as well as the implications of Epo upon the proliferation and survival of lung cancer cells. Since the protein expression of both Epo and EpoR is induced by hypoxia, which is frequently present in lung cancer, the cells were treated with Epo under both normoxic and hypoxic conditions (1% O2). By using quantitative (real-time) PCR, western blot analysis, and immunocytochemical staining, three non-small cell lung cancer (NSCLC) cell lines (A427, A549 and NCI-H358) were analyzed for the expression of EpoR and its specific downstream signaling pathways [Janus kinase2 (Jak2)-signal transducer and activator of transcription5 (STAT5), phosphatidylinositol-3-kinase (PI3K)-Akt, mitogen-activated protein (MAP) kinase]. The effects of 100U/ml Epo on cell proliferation and cisplatin-induced apoptosis were assessed. All NSCLC cell lines expressed EpoR mRNA and protein, while these levels differed considerably between the cell lines. We found the constitutive phosphorylation of EpoR and most of its downstream signaling pathways (STAT5, Akt and ERK1/2) independently of Epo administration. While Epo markedly enhanced the proliferation and reduced apoptosis of Epo-dependent UT-7/Epo leukemia cells, it did not affect tumor cell proliferation or the cisplatin-induced apoptosis of NSCLC cells. Thus, this invitro study suggests that there are no tumor-promoting effects of Epo in the NSCLC cell lines studied, neither under normoxic nor under hypoxic conditions.

Significance of Erythropoietin Receptor Antagonist EMP9 in Cancers.

We have clarified that cancer cells express their own erythropoietin (Epo) and its receptor (EpoR) mRNA levels, and the respective proteins, which are under the control of Epo-EpoR signaling. Then we explored to inhibit the Epo-EpoR signaling with an EpoR antagonist Epo mimetic peptide 9 (EMP9) that is a derivative of an Epo-mimicking peptide EMP1. In the study of the cancer cell lines in vitro, rhEpo accelerated the cancer cell growth, whereas the EMP9 inhibited the cell growth along with the inhibition of STAT5 tyrosine phosphorylation. Moreover, in vitro study of surgically resected histoculture of lung cancers revealed that EMP9 diminishes the expression of myoglobin in the cancer cells and destroys the feeding vessels. Additionally, in the xenografts of lung cancer histoculture, the EMP9 destroyed the xenografts by inducing apoptosis and suppressing proliferation of cancer cells in concomitant with macrophage accumulation. Furthermore, two types of perforations were detected in their cytoplasm: the one is mediated by nNOS in the cancer cells and the other one is by iNOS in the innate immune cells. These findings suggest that the inhibition of the Epo-EpoR signaling by EMP9 induces the cancer cell death that is mediated by the apoptosis and calcification of the cancer cells as well as the oxygen deficiency through the feeding vessels. Taken together, EMP9-based therapy may be a promising strategy to treat cancer patients.

Erythropoietin does not activate erythropoietin receptor signaling or lipolytic pathways in human subcutaneous white adipose tissue in vivo.

BackgroundErythropoietin (Epo) exerts direct effects on white adipose tissue (WAT) in mice in addition to its erythropoietic effects, and in humans Epo increases resting energy expenditure and affect serum lipid levels, but direct effects of Epo in human WAT have not been documented. We therefore investigated the effects of acute and prolonged Epo exposure on human WAT in vivo.MethodData were obtained from two clinical trials: 1) acute Epo exposure (rHuEpo, 400 IU/kg) followed by WAT biopsies after 1 h and 2) 10 weeks treatment with the erythropoiesis-stimulating agent (ESA) Darbepoietin-alpha. Biopsies were analyzed by PCR for Epo receptor (Epo-R) mRNA. A new and highly specific antibody (A82, Amgen) was used to evaluate the presence of Epo-R by western blot analysis in addition to Epo-R signaling proteins (Akt, STAT5, p70s6k, LYN, and p38MAPK), activation of lipolytic pathways (ATGL, HSL, CGI-58, G0S2, Perilipin, Cidea, Cidec, AMPK, and ACC), and mitochondrial biogenesis (VDAC, HSP90, PDH, and SDHA).ResultsNo evidence of in vivo activation of the Epo-R in WAT could be documented despite detectable levels of Epo-R mRNA.ConclusionThus, in contradiction to animal studies, Epo treatment within a physiological relevant range in humans does not exert direct effects in a subcutaneous WAT.

Erythropoietin accelerates tumor growth through increase of erythropoietin receptor (EpoR) as well as by the stimulation of angiogenesis in DLD-1 and Ht-29 xenografts.

Anemia is a relatively common symptom coexisting with colorectal carcinoma. Besides having a positive impact on hematological parameters, erythropoietin (Epo) has the serious adverse effect of promoting the neoplastic process. The role of Epo in colon cancer has not been clearly shown. The aim of this study was to assess the effects of Epo therapy on colorectal carcinoma cells both in in vitro and in animal models. Human colon adenocarcinoma cells DLD-1 and Ht-29 were cultured in medium with Epo beta in normoxia. Cell proliferation was measured with an automated cell counter. Expression of erythropoietin receptor (EpoR) mRNA, Akt mRNA, and their proteins were assessed by RT-PCR and confocal microscopy, respectively. Nude mice were inoculated with adenocarcinoma cells and treated with a therapeutic dose of Epo. Expression of EpoR, VEGF, Flt-1 and CD31 was evaluated in xenograft tumors. We identified that Epo through EpoR activates Akt, which promotes colon cancer cell growth and proliferation. Epo, and high levels of phosphorylated EpoR, directly accelerates tumor growth through its proliferative and proangiogenic effects. This study demonstrated that Epo had enhanced carcinogenesis through increase of EpoR and Flt-1 expression, and thereby contributed to tumor development. These results suggest that both EpoR-positive and EpoR-negative cancer cells could be regulated by exogenous Epo. However, an increased response to erythropoietin was observed in the EpoR-positive cells. Thus, erythropoietin increases the risk of tumor progression in colon cancer and should not be used to treat anemia in this type of cancer.

Influence of GATA-1 on Expression of EpoR in Bone Marrow CD71+ Cells of Rat Model with High Altitude Polycythemia

To explore the influence of GATA-1 on expression of EpoR in bone marrow CD71+ cells of rat model with high altitude polycythemia (HAPC). Forty-eight male SD rats were randomly divided into normal control and HAPC model group. HAPC model was established at the altitude of 4 300 meters in the natural environment, and verified by bone marrow cell counts and hematological parameters. Myeloid CD71+ cells were separated by the density gradient centrifugation combined with magnetic activated cell sorting. The expression of EpoR on cell membrane was detected by flow cytometry and cell immunofluorescence. The expression changes of GATA-1 and EpoR mRNA and protein were detected by Q-PCR and Western blot, respectively. CD71+ cells were cultured under normoxia and hypoxia, respectively. After transfection for 96 h, the optimal interference sequence GATA-1 shRNA1 was selected. And the mRNA and protein expression level of GATA-1 and EpoR were detected by Q-PCR and Western blot respectively. The animal model with HAPC was established successfully and comfirmed by the bone marrow cell counting and the hematologic parameters in comparison with that of the normal control. EpoR expression on the myeloid CD71+ cell membrane in HAPC group was significantly higher than that in normal control (P<0.05). The expression of GATA-1 and EpoR in myeloid CD71+ cells of HAPC group was higher than that in control group (P<0.05). The mRNA and protein expression of GATA-1 and EpoR in two groups positively correlated (control group, r=0.929, P<0.01, r=0.802, P<0.05; HAPC group, r=0.822, P<0.05, r=0.839, P<0.01). However, the mRNA and protein expression of EpoR at normoxia and hypoxia was significantly lower than that in negative control group after interfernce with GATA-1 shRNA1 for 96 h (P<0.05). And the expression of GATA-1 and EpoR under hypoxia was higher than that in normoxia. The effect of GATA-1 on EpoR expression may be correlated with the pathogenesis of HAPC.

Satellite cell response to erythropoietin treatment and endurance training in healthy young men.

Erythropoietin (Epo) treatment may induce myogenic differentiation factor (MyoD) expression and prevent apoptosis in satellite cells (SCs) in murine and in vitro models. Endurance training stimulates SC proliferation in vivo in murine and human skeletal muscle. In the present study, we show, in human skeletal muscle, that treatment with an Epo-stimulating agent (darbepoetin-α) in vivo increases the content of MyoD(+) SCs in healthy young men. Moreover, we report that Epo receptor mRNA is expressed in adult human SCs, suggesting that Epo may directly target SCs through ligand-receptor interaction. Moreover, endurance training, but not Epo treatment, increases the SC content in type II myofibres, as well as the content of MyoD(+) SCs. Collectively, our results suggest that Epo treatment can regulate human SCs in vivo, supported by Epo receptor mRNA expression in human SCs. In effect, long-term Epo treatment during disease conditions involving anaemia may impact SCs and warrants further investigation. Satellite cell (SC) proliferation is observed following erythropoitin treatment in vitro in murine myoblasts and endurance training in vivo in human skeletal muscle. The present study aimed to investigate the effects of prolonged erythropoiesis-stimulating agent (ESA; darbepoetin-α) treatment and endurance training, separately and combined, on SC quantity and commitment in human skeletal muscle. Thirty-five healthy, untrained men were randomized into four groups: sedentary-placebo (SP, n = 9), sedentary-ESA (SE, n = 9), training-placebo (TP, n = 9) or training-ESA (TE, n = 8). ESA/placebo was injected once weekly and training consisted of ergometer cycling three times a week for 10 weeks. Prior to and following the intervention period, blood samples and muscle biopsies were obtained and maximal oxygen uptake (V̇O2, max) was measured. Immunohistochemical analyses were used to quantify fibre type specific SCs (Pax7(+)), myonuclei and active SCs (Pax7(+)/MyoD(+)). ESA treatment led to elevated haematocrit, whereas endurance training increased V̇O2, max. Endurance training led to an increase in SCs associated with type II fibres (P < 0.05), whereas type I fibres showed no changes. Both ESA treatment and endurance training increased Pax7(+)/MyoD(+) cells, whereas only ESA treatment increased the total content of MyoD(+) cells. Epo-R mRNA presence in adult SC was tested with real-time RT-PCR using fluorescence-activated cell sorting (CD56(+)/CD45(-)/CD31(-)) to isolate cells from a human rectus abdominis muscle and was found to be considerably higher than in whole muscle. In conclusion, endurance training and ESA treatment may separately stimulate SC commitment to the myogenic program. Furthermore, ESA-treatment may alter SC activity by direct interaction with the Epo-R expressed on SCs.

Erythropoietin Mediated Increase in Monocyte-Derived Liver Macrophages; A Role for Kupffer Cells?

Erythropoietin (EPO) is the major hormone that drives mammalian erythropoiesis, via its surface receptor, EPO-R. It is mainly used for treating anemia associated with chronic renal failure and certain malignancies, although this latter indication is currently disputed. EPO-Rs were also found in non-erythroid cells, including dendritic cells and bone marrow macrophages (Lifshitz, 2008; 2010). Here we addressed the effect of EPO on hepatic-macrophages, namely resident liver macrophages (Kupffer cells) and liver monocyte-derived macrophages (MFs). Utilizing the rat Kupffer cell line (RKC-2) we demonstrate that these cells express EPO-R transcripts and cell surface EPO-R, as detected by our novel EPO-R antibody (GM1012; Maxwell 2015). EPO treatment of the RKC-2 cells led to a 1.5±0.06 fold increase (p<0.05) in EPO-R mRNA levels and a 2±0.01 fold decrease (p<0.01) in surface EPO-R levels. Stimulation of the cells with EPO induced a 15%±0.06 (p=0.01) increase in transcript levels of CCL-2 (a chemo attractant for monocytes) and a 15%±0.6 (p<0.05) increase in the levels of the secreted chemokine. EPO treatment also enhanced cellular activity of the RKC-2 cells as manifested in a 50%±0.13 (p<0.01) increase in cell migration, an increase in phagocytosis of microbeads (40%±0.08, p<0.01) and of E.coli (13%±0.05, p<0.01).Finally, in vivo experiments in which C57BL/6 mice were injected 3 times a week with 180U recombinant Human EPO (rHuEPO), demonstrated an EPO-induced selective increase in MFs (19.5%±0.01, p=0.05), but not in Kupffer cells. Elevated CCL-2 in sera of EPO-injected mice (2.1 fold increase, p<0.01) supports a mechanism by which EPO stimulates Kupffer cells to increase secretion of CCL-2, which in turn enhances recruitment of monocytes to the liver and their subsequent differentiation into MFs.The present study points to a new as hitherto unexplored action of EPO on two separate liver macrophage populations (Kupffer cells and MFs) which play crucial and distinct roles in liver homeostasis and immunity as well as in liver pathologies. EPO selective actions on liver macrophages thus call for future studies on the effects of this hormone on inflammation in the liver with a therapeutic goal to enhance liver immunity. DisclosuresMittelman:Celgene: Research Funding, Speakers Bureau; GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding, Speakers Bureau; Novartis Pharmaceuticals Corporation: Research Funding; Roche: Research Funding; Amgen: Research Funding.

Signals mediating Klotho-induced neuroprotection in hippocampal neuronal cells

The erythropoietin (Epo) receptor (EpoR) is expressed in the brain and was shown to have neuroprotective effects against brain damage in animal models. A recent study indicated that EpoR and its activity are the downstream effectors of Klotho for cytoprotection in the kidney. Thus, we propose that Klotho can stimulate the expression of EpoR in neuronal cells to enhance Epo-mediated protection. H19-7 hippocampal neuronal cells were treated with recombinant Klotho. In H19-7 cells, Klotho increased the expression of both the EpoR protein and mRNA. Klotho also enhanced the transcription activity of the EpoR promoter in H19-7 cells. Moreover, Klotho augmented the Epo-triggered phosphorylation of Jak2 and Stat5 and protected H19-7 cells from hydrogen peroxide cytotoxicity. The silencing of EpoR abolished the protective effect of Klotho against peroxide-induced cytotoxicity. Finally, the silencing of GATA1 diminished the Klotho-induced increase in EpoR protein and mRNA expression as well as its promoter activity. In conclusion, Klotho increased EpoR expression in neuronal cells through GATA1, thereby enabling EpoR to function as a cytoprotective protein against oxidative injury.

Prolonged erythropoietin treatment does not impact gene expression in human skeletal muscle.

We tested for the presence of erythropoietin receptor (Epo-R) in human skeletal muscle and alterations in gene expression after prolonged use of an erythropoiesis-stimulating agent (ESA). Nine healthy men were treated with ESA for 10 weeks (darbepoietin alfa). Muscle biopsies were collected before and after treatment. Alterations in gene expression were evaluated by gene array. Western blot and PCR analysis were used to test for Epo-R presence in human skeletal muscle. Very low Epo-R mRNA levels were found, but a new and sensitive antibody did not identify Epo-R protein in human skeletal muscle. The between-subject variation in skeletal muscle gene expression was greater than that observed in response to prolonged ESA treatment. Erythropoietin is unlikely to exert direct effects in human skeletal muscle due to a lack of Epo-R protein. Furthermore, prolonged ESA treatment does not seem to exert either direct or indirect effects on skeletal muscle gene expression.

Liver Macrophages (Kupffer cells) As Novel Targets of Erythropoietin

Erythropoietin (EPO) synthesized by the kidney and by the fetal liver is the major hormone that drives mammalian erythropoiesis. It is thus used in treatment of anemia, associated with chronic renal failure, as well as for alleviating chemotherapy or radiotherapy induced anemia in certain cancer patients. The physiological role of EPO is to regulate production of red blood cells via its receptor, EPO-R. However, EPO-R was found also in various other cell types, as shown also by us, suggesting that EPO may have pleiotropic activities. Originally, we have shown EPO effects on bone-marrow derived macrophages. We now report on EPO effects on liver macrophages. Using transgenic mice, overexpressing human EPO (tg6 mice), we found that they have a 2 fold increased level of F4/80 positive liver macrophages, compared to wt mice (p=0.01). In addition, C57bl/6 wt mice, injected 3 times a week with 180 U/ml of recombinant human EPO (rHuEPO) displayed a 2 fold increase in the percentage of F4/80 expressing cells (p=0.02), and in double positive F4/80 and CD11b expressing cells (p=0.03), compared to their untreated littermates. Similar results were observed in an experimental murine model of multiple myeloma (5T33). Hence, 5T33 MM mice that were treated with rHuEPO (10 consecutive days of 30 U/ml followed by 3 weekly injections of 30 U/ml for 2 weeks), presented a 2.5 fold increase (p=0.0004) and a 3 fold increase (p<0.0001) in the percentage of cells positive for F4/80 and in the percentage of cells double positive for F4/80 and CD11b, respectively. In addition, these mice displayed a 20% increase in the mean fluorescence intensity of CD11b, indicating that EPO causes not only an increase in the proportion of Kupffer cells, but it also induces the activation of CD11b positive cells, as reflected by the elevated expression of this marker per cell. We thus questioned whether Kupffer cells are direct targets of EPO. Our results show that the rat Kupffer cell line (KCL3-2) expresses EPO-R mRNA and protein on the cell surface, as detected by the novel EPO-R antibody, GM1012 generated by the FP7-Health European commission EpoCan grant (Maxwell et al., British Journal of Haematology, accepted for publication). Following treatment of the cells with EPO, the levels of EPO-R mRNA increased by 1.5 fold (p=0.02) and surface EPO-R levels decreased by 2 fold (p≤0.01). Furthermore, stimulation of the KCL3-2 cells with EPO led to activated MAPK signaling, and induced a 50% increase (p<0.005) in cell migration. A 3 fold increase (p=0.04) in the levels of the CCL-2 chemokine mRNA following treatment with EPO, points to a possible mechanism by which monocytes are recruited to the liver, where they differentiate into liver macrophages. Our findings thus introduce and elucidate a new function of EPO, focused on the liver, and indicate its effects in normal as well as in pathological conditions.The first two authors contributed equally to this study.This work was supported by the FP7-Health European commission EpoCan grant (282551). DisclosuresNo relevant conflicts of interest to declare.

Novel Evidence That Neuroblastoma and Rhabdomyosarcoma, Two Types of Small Round Blue Cell Tumors, Frequently Infiltrate Bone Marrow and Express Functional Erythropoietin Receptor (EpoR)—therapeutic Implications

Background: The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors, and recently our group demonstrated the presence of functional EpoR on human and murine germline-derived cell lines, including teratocarcinomas and ovarian cancer cells (J Ovarian Res. 2014 Jun 17;7:66). Neuroblastoma (NRB) and rhabdomyosarcoma (RMS), the most common soft-tissue sarcomas of adolescents and children and described as “small round blue cell tumors”, frequently infiltrate the BM to such a degree that they mimic acute lymphoblastic leukemia. These tumors express several cancer testis antigens (CTAs), which are characteristic of germline-derived cells. This is intriguing because of the 150-year-old “embryonic rest hypothesis of cancer development” by Rudolf Virchow and Julius Conheim, in which malignancies may develop from dormant embryonic/germ cells residing in adult tissues. Small round blue cell tumors, including in NRB and RMS, are potential candidates for such malignancies.Hypothesis. Based on our observations that germline-derived tumors express functional EpoR and that NRB and RMS express CTAs, we asked whether these pediatric sarcomas express functional EpOR as well.Materials and Methods. In our studies we employed four human NRB, eight RMS cell lines (4 embryonal-type RMS cell lines and 4 alveolar-type RMS cell lines), and mRNA samples obtained from patients. Expression of EpoR was evaluated by RT-PCR and FACS. The functionality of EpoR in NRB and RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS and NRB cells were exposed to vincristine (VCR) in the presence or absence of EpO to test whether EpO may impair the therapeutic effect of VCR.Results. We found expression of EpoR mRNA in all tested cell lines and patient samples. Importantly, EpoR has been found to be functional in all NRB and RMS cell lines tested, responding to stimulation by erythropoietin (EPO) by an increase in chemotaxis, adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, we found that EPO stimulates proliferation of NRB and RMS cells and increases their resistance to VCR.Conclusions. Our data for the first time indicates that human pediatric sarcomas express functional EpoR. This finding has important clinical implications, as EPO is frequently employed to ameliorate chemotherapy-related anemia. The presence of functional EpoR on pediatric sarcoma cells indicates that EPO supplementation may have the unwanted side effect of tumor progression. Finally, the presence of functional EpoR on both NRB and RMS cells and germline-derived tumors as well as expression of CTA antigens by these cells supports the hypothesis that pediatric sarcomas may develop in developing tissues from embryonic remnants with germline potential. DisclosuresNo relevant conflicts of interest to declare.

Molecular and functional characterization of erythropoietin receptor of the goldfish (Carassius auratus L.)

Erythropoietin receptor (EPOR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and survival of erythrocyte progenitor cells. This study reports on the molecular and functional characterization of goldfish EPOR. The identified goldfish EPOR sequence possesses the conserved EPOR ligand binding domain, the fibronectin domain, the class I cytokine receptor superfamily motif (WSXWS) as well as several intracellular signaling motifs characteristic of other vertebrate EPORs. The expression of epor mRNA in goldfish tissues, cell populations and cells treated with recombinant goldfish EPO (rgEPO) were evaluated by quantitative PCR revealing that goldfish epor mRNA is transcribed in both erythropoietic tissues (blood, kidney and spleen) and non-hematopoietic tissues (brain, heart and gill), as well as in immature erythrocytes. Recombinant goldfish EPOR (rgEPOR), consisting of its extracellular domain, dose-dependently inhibited proliferation of progenitor cells induced by rgEPO. In vitro binding studies indicated that rgEPO exists as monomer, dimer and/or trimmer and that rgEPOR exists as monomer and/or homodimer, and when incubated together, formed a ligand–receptor complex. Our results demonstrate that goldfish EPO/EPOR signaling has been highly conserved throughout vertebrate evolution as a required mechanism for erythrocyte development.

The erythropoietin receptor is not required for the development, function, and aging of rods and cells in the retinal periphery.

Erythropoietin (EPO) was originally described for its antiapoptotic effects on erythroid progenitor cells in bone marrow. In recent years, however, EPO has also been shown to be cytoprotective in several tissues, including the retina. There, exogenous application of EPO was reported to exert neuro- and vasoprotection in several models of retinal injury. EPO and the erythropoietin receptor (EPOR) are expressed in the retina, but the role of endogenous EPO-EPOR signaling in this tissue remains elusive. Here, we investigated the consequences for cell physiology and survival when EpoR is ablated in rod photoreceptors or in the peripheral retina. Two mouse lines were generated harboring a cyclization recombinase (CRE)-mediated knockdown of EpoR in rod photoreceptors (EpoR(flox/flox);Opn-Cre) or in a heterogeneous cell population of the retinal periphery (EpoR(flox/flox);α-Cre). The function of the retina was measured with electroretinography. Retinal morphology was analyzed in tissue sections. The vasculature of the retina was investigated on flatmount preparations, cryosections, and fluorescein angiography. Retinal nuclear layers were isolated by laser capture microdissection to test for EpoR expression. Gene expression analysis was performed with semiquantitative real-time PCR. To test if the absence of EPOR potentially increases retinal susceptibility to hypoxic stress, the knockdown mice were exposed to hypoxia. Newborn mice had lower retinal expression levels of EpoR and soluble EpoR (sEpoR) than the adult wild-type mice. In the adult mice, the EpoR transcripts were elevated in the inner retinal layers, while expression in the photoreceptors was low. CRE-mediated deletion in the EpoR(flox/flox);Opn-Cre mice led to a decrease in EpoR mRNA expression in the outer nuclear layer. A significant decrease in EpoR expression was measured in the retina of the EpoR(flox/flox);α-Cre mice, accompanied by a strong and significant decrease in sEpoR expression. Analysis of the retinal morphology in the two knockdown lines did not reveal any developmental defects or signs of accelerated degeneration in the senescent tissue. Similarly, retinal function was not altered under scotopic and photopic conditions. In addition, EpoR knockdown had no influence on cell viability under acute hypoxic conditions. Retinal angiogenesis and vasculature were normal in the absence of EPOR. However, expression of some EPOR-signaling target genes was significantly altered in the retinas of the EpoR(flox/flox);α-Cre mice. Our data suggest that expression of EPOR in rod photoreceptors, Müller cells, and amacrine, horizontal, and ganglion cells of the peripheral retina is not required for the maturation, function, and survival of these cells in aging tissue. Based on the expression of the EPOR-signaling target genes, we postulate that expression of soluble EPOR in the retina may modulate endogenous EPO-EPOR signaling.

Expression and functional significance of the erythropoietin receptor in hepatocellular carcinoma

BackgroundErythropoietin, through its specific receptor (EpoR), may induce responses in a variety of non-haematopoietic tissues including malignant cells. The purpose of this study was to examine the expression of EpoR in hepatocellular carcinoma (HCC) and to correlate the levels of EpoR expression with the clinicopathological properties of HCC and tumour recurrence. MethodsThe study included 134 patients who underwent curative hepatectomy for hepatitis B virus (HBV)-related primary HCC. The clinical, laboratory and pathological data from these patients were retrospectively collected. The expression of EpoR mRNA and protein were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. ResultsExpression of EpoR mRNA in the cirrhotic liver was positively correlated with tumour cell differentiation and 1-year disease-free survival (74.8% in the high expression group versus 46.9% in the low expression group; P = 0.001), as it was for EpoR mRNA expression in HCC (64.4% in the high expression group versus 52.7% in the low expression group; P = 0.044). Tumour recurrence showed stronger dependence on the expression of EpoR protein in non-malignant cirrhotic livers than in HCC. ConclusionIn HBV-related HCC, the levels of EpoR mRNA and protein in non-tumour cirrhotic livers were positively correlated with tumour cell differentiation, which is a favourable predictor of disease-specific survival.

Mechanism of endurance training-induced erythrocyte deformability in rats involves erythropoiesis

Higher erythrocyte deformability may reduce the risk of circulatory diseases by enhancing oxygen delivery and reducing the load on the cardiovascular system. The effect of endurance training on erythrocyte deformability is not clear. This study explored the impact of endurance training on erythrocyte deformation and shape and investigated the underlying mechanisms of hemorheological alterations. Forty male Wistar rats were randomly divided into two groups: sedentary (S; n = 21) and exercised (E; n = 19). Hematological indices and erythrocyte shape were measured at the end of the 11th week. The gene expression of erythropoietin (Epo) and the Epo receptor (EpoR) was quantified using reverse transcription-PCR (RT-PCR), and Epo protein expression was analyzed using Western blotting. Endurance training significantly decreased the abnormality ratio of erythrocyte shape (P < 0.01). The deformability indicator (DI) of red blood cells was lower in the E group than in the S group (P < 0.01). Eleven weeks of endurance training increased Epo mRNA and protein expression in the kidney (P < 0.01), EpoR mRNA expression in the bone marrow (P < 0.05), and relative circulating Epo (P < 0.01) compared to the sedentary group. The decrease in the erythrocyte morphological index and the maximum of deformability indicator were associated with an increase in relative circulating Epo. In conclusion, 11 weeks of endurance training increased erythrocyte deformability. Epo and EpoR may contribute to the decreased morphological index and deformability indicator in erythrocytes during endurance training in rats.