Erythropoietin Receptor Signaling Research Articles

Abstract 2771: Increased mtDNA copy number via EPOR signaling: A potential mechanism for metastatic dissemination in head and neck squamous cell carcinoma

Abstract Introduction: Head and Neck Squamous cell carcinoma (HNSCC) is characterized by high degree of invasion, locoregional metastasis, relapse which leads to overall poor prognosis. The molecular mechanism responsible for aggressiveness is poorly understood. A better understanding of molecular pathways involved shall precede the identification of newer diagnostic tools and therapeutic targets. We explored EPOR signaling pathway in Circulating tumor cells (CTCs) and HNSCC cell lines. Methods and Results: By using a validated in-house CTC isolation protocol, which addresses various drawbacks of currently available techniques and isolates CTCs suitable for various downstream molecular analysis, we isolated CTCs from HNSCC patients. The whole transcriptome analysis was performed from CTCs using ultra-low cell number RNA sequencing and corresponding primary tumors in 24 paired samples. We found a significant transcriptomic heterogeneity in CTCs, which are associated with overall disease outcome in HNSCC patients. To explore the plausible molecular pathways, we also identified 521 differentially expressed genes in CTCs with respect to primary tumors and various canonical pathways including oxidative phosphorylation, TCA cycle, cytokine-cytokine receptor interaction signaling, and ECM receptor interaction were enriched in CTC samples. Overexpression of erythropoietin receptor (EPOR) along with various genes encoded by mitochondrial genome and genes involved in maintenance of mtDNA in CTCs as compared to primary tumors was an important finding from our data. When analyzed in depth, there was a significant positive correlation between various genes involved in mtDNA maintenance (ABAT, NT5M) and encoded by mitochondrial genome (MT-ND1, MT-ND2, MT-ND4, MT-ND5, MT-ND6, MT-CO1, MT-CO2, MT-ATP6, MT-CYTB) with EPOR expression in our RNA seq data which is also substantiated by analyzing TCGA data. We confirmed these findings experimentally in HNSCC cell lines Cal 27 and FaDu. It was also observed that metastatic cell line FaDu has significantly higher mtDNA content as compared to non-metastatic cell line Cal 27, which was correlated with EPOR expression. Treatment with low dose Erythropoietin (0.1IU/mL) which a natural ligand for EPOR, enhanced mtDNA content in both the cell lines at low doses and boosted the metastatic capability while minimal effect on cell proliferation was observed. Conclusion: EPOR signaling might promote the metastatic ability of cancer cells and may act as a potential target for cancer dissemination and metastasis. Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Increased mtDNA copy number via EPOR signaling: A potential mechanism for metastatic dissemination in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2771.

Erythropoietin receptor signal is crucial for periodontal ligament stem cell-based tissue reconstruction in periodontal disease

Alveolar bone loss caused by periodontal disease eventually leads to tooth loss. Periodontal ligament stem cells (PDLSCs) are the tissue-specific cells for maintaining and repairing the periodontal ligament, cementum, and alveolar bone. Here, we investigated the role of erythropoietin receptor (EPOR), which regulates the microenvironment-modulating function of mesenchymal stem cells, in PDLSC-based periodontal therapy. We isolated PDLSCs from patients with chronic periodontal disease and healthy donors, referred to as PD-PDLSCs and Cont-PDLSCs, respectively. PD-PDLSCs exhibited reduced potency of periodontal tissue regeneration and lower expression of EPOR compared to Cont-PDLSCs. EPOR-silencing suppressed the potency of Cont-PDLSCs mimicking PD-PDLSCs, whereas EPO-mediated EPOR activation rejuvenated the reduced potency of PD-PDLSCs. Furthermore, we locally transplanted EPOR-silenced and EPOR-activated PDLSCs into the gingiva around the teeth of ligament-induced periodontitis model mice and demonstrated that EPOR in PDLSCs participated in the regeneration of the periodontal ligament, cementum, and alveolar bone in the ligated teeth. The EPOR-mediated paracrine function of PDLSCs maintains periodontal immune suppression and bone metabolic balance via osteoclasts and osteoblasts in the periodontitis model mice. Taken together, these results suggest that EPOR signaling is crucial for PDLSC-based periodontal regeneration and paves the way for the development of novel options for periodontal therapy.

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence-guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease

AbstractDisordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon α (IFNα), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2–containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFNα-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFNα-CISH axis.

Using Inducible Signaling Receptors to Increase Erythropoietic Output from Genome-Edited Hematopoietic Stem Cells

Introduction: Oxygen is delivered to all tissues in the body through the hemoglobin molecule uniquely present in red blood cells (RBCs). Proper production and function of RBCs is therefore critical to human health. However, genetic defects of the hematologic system such as anemias and hemoglobin disorders (broadly termed “hemoglobinopathies”) are among the most common genetic disorders in the world. Therefore, developing strategies to regulate RBC development may allow researchers and clinicians to increase therapeutic potential of hematologic cell and gene therapies. Erythropoietin (EPO) signaling is indispensable to RBC development. In its native form, two EPO receptor (EPOR) monomers dimerize in the presence of EPO cytokine to initiate a downstream signaling cascade. Prior work has shown that conformational requirements of EPOR dimerization are rather “loose” and signaling may also be initiated by agonistic diabodies or in the context of chimeric receptors. While these chimeric receptors have limited clinical applicability, inducible dimerization domains such as engineered FKBPs are currently being used in clinical trials in the form of inducible safety switches. Due to the modular nature of cytokine receptors, we hypothesized that inducible dimerization domains (such as the FKBP domain) could be paired with endogenous receptors and signaling pathways to generate chimeric receptors that place RBC development under the control of a bioavailable small molecule - and therefore in the control researchers and clinicians. This study presents a set of chimeric, synthetic receptors that support HSPC output towards the erythroid lineage via a non-immunogenic, non-toxic small molecule (AP30187, hereafter termed “BB”). Methods: In-vitro editing was performed in primary human CD34+ HSPCs by electroporating Cas9 protein complexed with sgRNA along with an AAV6 DNA repair template to facilitate gene insertion via homology-directed repair (HDR). Bulk edited CD34+ HSPCs were then differentiated into RBCs using established differentiation protocols in the presence and absence of both erythropoietin and BB. Differentiation was determined by staining for RBC surface markers (CD71 and GPA) and subsequent flow cytometry analysis. Erythropoietic output was determined by cell density counts. Allele frequency of the edited cell population was determined through droplet digital PCR. Results: We generated seven FKBP-EPOR chimeric receptors - with the basic structure of SFFV-[FKBP-EPOR]-2A-YFP-bGH - that placed the FKBP binding domain at different regions of the endogenous receptor (Figure 1) and analyzed their ability to support RBC differentiation in the absence of EPO but with BB. Out of the seven constructs we found a set of receptors - 1.4 and 1.5 - that demonstrated erythropoietin-independent, BB-mediated erythroid differentiation at 108% and 94% mock RBC differentiation respectively. Construct 1.5 - dubbed inducible EPOR (iEPOR) - was chosen over 1.4 for further optimization because it was also no longer EPO responsive. We further optimized the design of iEPOR by 1) introducing a secretory signal peptide to improve trafficking of the receptor to the cell surface; 2) inclusion of a naturally occurring EPOR truncation that results in supraphysiological RBC differentiation; and 3) modification of the genomic insertion site and promoter of the iEPOR cassette to generate a toolbox of expression profiles. Our results suggest that iEPOR mimics the native EPOR signaling cascade with high fidelity and sensitivity according to bulk RNA-Seq data. We also observe a significant increase in erythropoietic output for iEPOR version 1.4 (6.7-fold by day 11 of RBC differentiation in +BB over -BB conditions, P=0.003), which was further enhanced by the above modifications for vector iEPOR version 3.5.2 (12.9-fold by day 11 of RBC differentiation in +BB over -BB conditions, P<0.0001). Additionally, iEPOR is demonstrated to be erythropoietin-independent, dose-responsive, and tunable via expression from different loci and promoters. Conclusion:iEPORs represent a powerful new tool to alter the cellular dynamics of red blood cell development. The work demonstrated in this study has the potential to dramatically improve the efficacies of existing gene therapies, offer a route to EPO-free ex-vivo RBC production, as well as serve as a new tool by which to better understand stem cell biology and differentiation.

Erythropoietin Receptor (EPOR) Signaling in the Osteoclast Lineage Contributes to EPO-Induced Bone Loss in Mice.

Erythropoietin (EPO) is a pleiotropic cytokine that classically drives erythropoiesis but can also induce bone loss by decreasing bone formation and increasing resorption. Deletion of the EPO receptor (EPOR) on osteoblasts or B cells partially mitigates the skeletal effects of EPO, thereby implicating a contribution by EPOR on other cell lineages. This study was designed to define the role of monocyte EPOR in EPO-mediated bone loss, by using two mouse lines with conditional deletion of EPOR in the monocytic lineage. Low-dose EPO attenuated the reduction in bone volume (BV/TV) in Cx3cr1Cre EPORf/f female mice (27.05%) compared to controls (39.26%), but the difference was not statistically significant. To validate these findings, we increased the EPO dose in LysMCre model mice, a model more commonly used to target preosteoclasts. There was a significant reduction in both the increase in the proportion of bone marrow preosteoclasts (CD115+) observed following high-dose EPO administration and the resulting bone loss in LysMCre EPORf/f female mice (44.46% reduction in BV/TV) as compared to controls (77.28%), without interference with the erythropoietic activity. Our data suggest that EPOR in the monocytic lineage is at least partially responsible for driving the effect of EPO on bone mass.

Role of Erythropoietin Receptor Signaling in Macrophages or Choroidal Endothelial Cells in Choroidal Neovascularization.

Erythropoietin (EPO) has been proposed to reduce the progression of atrophic age-related macular degeneration (AMD) due to its potential role in neuroprotection. However, overactive EPO receptor (EPOR) signaling increased laser-induced choroidal neovascularization (CNV) and choroidal macrophage number in non-lasered mice, which raised the question of whether EPOR signaling increased CNV through the recruitment of macrophages to the choroid that released pro-angiogenic factors or through direct angiogenic effects on endothelial cells. In this study, we addressed the hypothesis that EPOR signaling increased CNV by direct effects on macrophages or endothelial cells. We used tamoxifen-inducible macrophage-specific or endothelial cell-specific EPOR knockout mice in the laser-induced CNV model, and cultured choroidal endothelial cells isolated from adult human donors. We found that macrophage-specific knockout of EPOR influenced laser-induced CNV in females only, whereas endothelial-specific knockout of EPOR reduced laser-induced CNV in male mice only. In cultured human choroidal endothelial cells, knockdown of EPOR reduced EPO-induced signal transducer and activator of transcription 3 (STAT3) activation. Taken together, our findings suggest that EPOR signaling in macrophages or choroidal endothelial cells regulates the development of CNV in a sex-dependent manner. Further studies regarding the role of EPO-induced signaling are required to assess EPO safety and to select or develop appropriate therapeutic approaches.

P1597: THE DYNAMIC INTERACTION BETWEEN INFLAMMATION AND IRON SENSORS IN MALARIA-INDUCED ANEMIA

Background: Severe malarial anemia (SMA) is a complication developed during Plasmodium infection. It is often associated with increased rates of malaria-related morbidity and mortality, especially in children and pregnant women. Hence, it is fundamental to investigate new parameters to prevent this lethality. Aims: The occurrence of ineffective erythropoiesis, caused by a dysregulated erythropoietin (EPO)-erythropoietin receptor (EPOR) signaling pathway, is one of the main features of SMA. Therefore, to gain new insights into the pathogenesis of SMA, our study aims to better understand the dynamic interaction between erythropoiesis and inflammation in mice exposed to single or multiple infections, mimicking what occurs in endemic regions. Methods: Erythroid cell differentiation and immune cells activation were evaluated by flow cytometry. Changes in iron metabolism were assessed by the differences in expression of genes regulating iron homeostasis by qRT-PCR. Results: Flow cytometry analyses suggested that additional exposure to the parasite enhances erythropoiesis. Comparing infected with reinfected mice, we observed a faster erythropoietic response, induced by the bone marrow. It was also found that reinfected mice presented an earlier and robust immune response activation, as developing innate immune memory. Due to the fundamental role of iron in erythropoiesis and immune response activation, the expression of iron sensors, like HFE, was analyzed, detecting a strict correlation between disease severity and multiple infections exposure. The action of HFE in the pathogenesis of SMA and its role on the immune response activation, elicited to combat malaria, was also investigated. Preliminary data shows that when HFE knockout mice were infected with the parasite, their anemic profile improves. Summary/Conclusion: These reveals HFE as a potential target for drug therapy during hemolytic disorders, such as malaria infection.

Vhl deficiency in Dmp1-expressing cells affects myeloid development and erythropoiesis: possible effects on myeloerythroid metabolism

Abstract The specific types of bone marrow (BM) stromal cells and local BM microenvironments that contribute to the regulation of myelopoiesis are not fully understood. The von-Hippel Lindau protein (VHL) controls responses to hypoxia and could regulate immune metabolism. We investigated this in Dmp1-Cre; Vhl conditional knockout mice (cKO), where Vhl is deleted in mesenchymal stem cells, osteoblasts (OBs) and osteocytes. Similar to previous findings when Vhl is deleted in OBs, we observed low blood glucose levels in the cKO mice, which may reflect an increase in OB cellular glycolysis and increased glucose uptake in the bone. Erythropoietin (Epo) treatment in vivo reduced blood glucose levels in rats. In preliminary studies, we found elevated Epo levels in cKO peripheral blood serum and BM fluid by 6 weeks of age, and evidence for dysregulated erythropoiesis. Moreover, the cKO displayed an increased frequency of common myeloid progenitors, CD11b+ Gr1− monocytes, and CD11b+ Gr1+ granulocytes by 10 weeks of age. We hypothesize that alterations in skeletal glucose metabolism directly affect myeloerythroid development in the BM through increased Epo-receptor (EpoR) signaling. EpoR is expressed on osteoprogenitors, hematopoietic stem cells, and B cells, but whether it is expressed on myeloid progenitors is unclear. Studies are in progress to test the hypothesis that overproduction of Epo by Vhl-deficient Dmp1+ cells results in elevated EpoR signaling in myeloid progenitors and lineages; this in turn, results in an increase in myeloid cell glycolysis and Glut1 expression and proliferation. These studies are relevant for the understanding how BM microenvironmental changes can trigger adaptations in immunometabolism of myeloid cells. Supported by grants from NIH (R15 AI154245-01, F31 AI154815), and University of California, Merced

Human constitutive androstane receptor represses liver cancer development and hepatoma cell proliferation by inhibiting erythropoietin signaling

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO–EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal–regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR–hepatocyte nuclear factor 4 alpha–EPO axis in human liver tumorigenesis.

Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a condition with an imbalanced inflammatory response and delayed resolution of inflammation. Macrophage polarization plays an important role in inflammation and resolution. However, the mechanism of macrophage polarization in ALI/ARDS is not fully understood. We found that mice with lipopolysaccharide administration developed lung injury with the accumulation of extracellular cold-inducible RNA-binding protein (eCIRP) in the lungs. eCIRP, as a damage-associated molecular pattern (DAMP), inhibited M2 macrophage polarization, thereby tipping the balance toward inflammation rather than resolution. Anti-CIRP antibodies reversed such phenotypes. The levels of macrophage erythropoietin (EPO) receptor (EPOR) were reduced after eCIRP treatment. Myeloid-specific EPOR-deficient mice displayed restrained M2 macrophage polarization and impaired inflammation resolution. Mechanistically, eCIRP impaired Rab26, a member of Ras superfamilies of small G proteins, and reduced the transportation of surface EPOR, which resulted in macrophage polarization toward the M1 phenotype. Moreover, EPO treatment hardly promotes M2 polarization in Rab26 knockout (KO) macrophages through EPOR. Collectively, macrophage EPOR signaling is impaired by eCIRP through Rab26 during ALI/ARDS, leading to the restrained M2 macrophage polarization and delayed inflammation resolution. These findings identify a mechanism of persistent inflammation and a potential therapy during ALI/ARDS.

The Non-Erythropoietic EPO Analogue (Cibinetide) Preserves Bone Mass in Mice

Background and aims: Erythropoietin (EPO) is a pleiotropic cytokine, which besides its classical role in driving erythropoiesis, displays tissue protective and immunomodulatory activities. EPO also induces bone loss. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. Cibinetide (CIB), a non-erythropoietic analogue of EPO, specifically binds to the heteromeric receptor and confers tissue protection. Our published findings that EPO stimulates osteoclast precursors and entrains a decrease in bone density, raise questions regarding the underlying molecular mechanisms. Here, we evaluated the role of the heteromeric complex in bone metabolism using CIB alone and in combination with EPO in vivo and in vitro.Results: CIB injections to 12-week-old female mice (120 µg/kg thrice weekly for 4 weeks) resulted in a significant increase in tissue mineral density in cortical bone by 5.8% (1416.4±39.27 vs 1338.74±16.56 mgHA/cm 3) and in trabecular bone by 5.2% (1056.52±30.94 vs 1004.13±16.91 mg HA/cm 3) (n=10 in each group, p< 0.05 versus saline-injected controls), as measured by microCT (Figure 1A).To evaluate the capacity of CIB to attenuate EPO mediated bone loss, we administered CIB (300 µg/kg) for 5 consecutive days, to 13-week-old female mice that also received 2 injections of 120U EPO on days 1 and 4. Flow cytometry analysis revealed a 1.8-fold reduction in the number of osteoclast progenitors, defined as Lin -CD11b −CD115 +Ly6C hi, in the EPO + CIB injected mice, compared to the mice injected with EPO alone (n=7 in each group, p< 0.05). Hemoglobin levels and TER119 + bone marrow (BM) erythroid progenitors were similar in both groups.In vitro, EPO administration to BM-derived macrophages (BMDM) enhanced osteoclastogenesis, whereas CIB had an opposite, dose-dependent effect. Combining CIB with EPO inhibited osteoclastogenesis in BMDM, suggesting that CIB overrides the pro-osteoclastogenic effect of EPO (Figure 1B).Conclusions: Our findings highlight the increasing complexity of EPOR signaling in bone and pave the way for clinical translation through potential combination therapy of EPO and CIB in anemic and in cancer patients. Adjunctive administration of CIB may prevent or attenuate bone loss while preserving the erythropoietic actions of EPO.This study was supported by a grant from the Dotan Hemato-oncology Fund, the Cancer Biology Research Center, Tel Aviv University to DN and YG. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Erythropoietin (EPO) as a Key Regulator of Erythropoiesis, Bone Remodeling and Endothelial Transdifferentiation of Multipotent Mesenchymal Stem Cells (MSCs): Implications in Regenerative Medicine.

Human erythropoietin (EPO) is an N-linked glycoprotein consisting of 166 aa that is produced in the kidney during the adult life and acts both as a peptide hormone and hematopoietic growth factor (HGF), stimulating bone marrow erythropoiesis. EPO production is activated by hypoxia and is regulated via an oxygen-sensitive feedback loop. EPO acts via its homodimeric erythropoietin receptor (EPO-R) that increases cell survival and drives the terminal erythroid maturation of progenitors BFU-Es and CFU-Es to billions of mature RBCs. This pathway involves the activation of multiple erythroid transcription factors, such as GATA1, FOG1, TAL-1, EKLF and BCL11A, and leads to the overexpression of genes encoding enzymes involved in heme biosynthesis and the production of hemoglobin. The detection of a heterodimeric complex of EPO-R (consisting of one EPO-R chain and the CSF2RB β-chain, CD131) in several tissues (brain, heart, skeletal muscle) explains the EPO pleotropic action as a protection factor for several cells, including the multipotent MSCs as well as cells modulating the innate and adaptive immunity arms. EPO induces the osteogenic and endothelial transdifferentiation of the multipotent MSCs via the activation of EPO-R signaling pathways, leading to bone remodeling, induction of angiogenesis and secretion of a large number of trophic factors (secretome). These diversely unique properties of EPO, taken together with its clinical use to treat anemias associated with chronic renal failure and other blood disorders, make it a valuable biologic agent in regenerative medicine for the treatment/cure of tissue de-regeneration disorders.

Monotcytes/Macrophages Are Involved in Erythrocytosis Caused By Both EPO-Dependent and -Independent Activation of EPOR

Excess activation of EPO receptor (EPOR) signaling leads to erythrocytosis, which is defined as an absolute increase of red cell mass. EPO-independent and erythrocytes-intrinsic EPOR activation is induced by gain-of-function EPOR or JAK2 mutations, resulting in “primary” erythrocytosis. Erythrocytes-extrinsic EPOR activation is called “secondary” erythrocytosis, which is caused by EPOR stimulation with high level of EPO.In order to investigate primary and secondary erythrocytosis in detail, we generated two unique mouse models. LysM -Cre; Jak2 V617F knock-in heterozygous mice express constitutively active JAK2 in LysM-expressing cells, which include a small portion of HSCs and their downstream progenitors. We also generated a Alb -Cre; Rosa26 LoxP-Stop-LoxP-rtTA (LSL-rtTA);human EPAS1 double-point-mutant (DPM) mice (Alb -Cre;DPM). EPAS1 is a master regulator of EPO gene. The EPAS1-DPM has mutations in P531 and N847 sites, which are critical sites for stability and transcriptional inactivation in normoxia. This mouse expresses the DPM mutant (P531A and N847A) specifically in hepatocytes in a doxycycline-inducible manner, resulting in high EPO production in the liver. We used LysM -Cre; Jak2 V617F as an erythrocytes-intrinsic “primary” erythrocytosis model, and Alb -Cre;DPM as an erythrocytes-extrinsic “secondary” erythrocytosis model. Interestingly, both of them developed comparable degree of erythrocytosis quickly. The plasma EPO level in LysM -Cre; Jak2 V617F was significantly decreased, while that in Alb -Cre/DPM was significantly increased in comparison to the control and also to LysM -Cre; Jak2 V617F. They both exhibited splenomegaly, but spleens of Lys M-Cre; Jak2 V617F mice were significantly larger than those of Alb -Cre;DPM mice. In Alb -Cre;DPM mice, CD45- CD71+ erythroblasts were detected in the liver, whereas there were almost no erythroblasts in the liver of the control mice, suggesting that extremely high level of EPO expression in the liver causes erythropoiesis in the liver of Alb -Cre;DPM mice.Increased monocyte count has been reported in patients with erythrocytosis. Consistent with the observation in the patients, total CD11b+ CD115+ monocytes were significantly increased in PB from both LysM -Cre; Jak2 V617F and Alb -Cre;DPM mice compared to those in PB from the wild-type mice. It is also well known that macrophages play a crucial role in erythropoiesis, supplying the iron and engulfing the protruded nuclei from erythroblasts in the terminal maturation process. The complex of erythroblasts and macrophages directly interacting each other is called as “erythroblastic island (EBI)”. Thus, we next analyzed macrophages in bone marrow (BM) and spleen (SP) in our erythrocytosis models. Macrophages in SP were dramatically increased in the both models, while those in BM were not significantly altered. The percentage of EBI (multiplet F4/80+ Ter119+) in BM and SP were increased in both models.EPOR expression on the cell surface is required for EPO-EPOR signaling. To confirm the expression of EPOR by genetic approach, we combined EPOR -Cre knock-in allele and Rosa26LSL-GFP reporter allele to generate EPOR -Cre;GFP reporter mice. In this model, pro-erythroblasts and basophilic erythroblasts in BM expressed GFP as expected. We did not find GFP expression on the Macrophage and dendritic cell precursors (MDPs), common monocyte progenitors (cMoPs), mature monocytes in BM and PB monocytes. In a single-cell-gated macrophage population, we found a small fraction expressing GFP. However, FACS sorting revealed that those GFP+ cells are not macrophages alone, but macrophages with erythroblasts adhered to them. Even after adding EDTA, some of those macrophages still retain bound erythroblasts. It is possible that they could acquire activated EPOR signaling from erythroblasts within EBI through direct interaction. We are currently investigating the mechanisms for expansion of monocytes/macrophages in erythrocytosis.Collectively, here we demonstrate that both Jak2V617F and high EPO could activate EPOR signaling and expand erythroblasts and monocytes/macrophages, which might contribute to erythrocytosis phenotype in our primary and secondary erythrocytosis models. Further exploration of the roles of monocytes/macrophages in erythrocytosis could lead to development of novel therapeutic strategy for erythrocytosis by modulating their functions in EBI. DisclosuresNo relevant conflicts of interest to declare.

Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoproteincytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.

Epo receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation

AbstractAnemic stress induces stress erythropoiesis, which rapidly generates new erythrocytes to restore tissue oxygenation. Stress erythropoiesis is best understood in mice where it is extramedullary and occurs primarily in the spleen. However, both human and mouse stress erythropoiesis use signals and progenitor cells that are distinct from steady-state erythropoiesis. Immature stress erythroid progenitors (SEPs) are derived from short-term hematopoietic stem cells. Although the SEPs are capable of self-renewal, they are erythroid restricted. Inflammation and anemic stress induce the rapid proliferation of SEPs, but they do not differentiate until serum erythropoietin (Epo) levels increase. Here we show that rather than directly regulating SEPs, Epo promotes this transition from proliferation to differentiation by acting on macrophages in the splenic niche. During the proliferative stage, macrophages produce canonical Wnt ligands that promote proliferation and inhibit differentiation. Epo/Stat5-dependent signaling induces the production of bioactive lipid mediators in macrophages. Increased production of prostaglandin J2 (PGJ2) activates peroxisome proliferator-activated receptor γ (PPARγ)-dependent repression of Wnt expression, whereas increased production of prostaglandin E2 (PGE2) promotes the differentiation of SEPs.

Erythropoietin Receptor Signaling Supports Retinal Function after Vascular Injury

The investigation of erythropoietin (EPO) has expanded to include potential nonhematopoietic roles in neural and retinal diseases, including diabetic retinopathy. However, it remains unclear how EPO functions to support the neural retina. Transgenic mice with hypoactive EPO receptor (EPOR) signaling (hWtEPOR) were compared with littermate control mice (WT) to test the role of EPOR signaling under normal conditions and after vascular injury and regrowth into the retina. Although retinal function tested with OptoMotry and electroretinography was comparable to adult (8-week-old) littermate WT mice, hWtEPOR mice had thinner inner and outer plexiform layers and a greater number of amacrine cells. Injury and repair caused by the oxygen-induced retinopathy model reduced visual acuity thresholds, reduced electroretinography amplitudes, and thinned the outer plexiform and inner nuclear layers of both WT and hWtEPOR 8-week-old mice. In hWtEPOR compared with WT mice, scotopic a-wave amplitudes were reduced by injury, despite no change in outer nuclear layer thickness; and peripheral rod, but not cone number, was reduced. Scotopic b-waves were reduced in injured hWtEPOR mice compared with WT, and rod bipolar cell ectopic neurites were increased in both genotypes after injury, suggesting a potential reparative process to preserve connectivity and the b-wave. Normal EPOR signaling appeared important because ectopic neurites and b-waves were lower in the hWtEPOR than WT injured mice.

Erythropoietin attenuates vascular calcification by inhibiting endoplasmic reticulum stress in rats with chronic kidney disease

Previous studies suggested that endoplasmic reticulum (ER) stress induced-apoptosis promoted vascular calcification (VC). Interestingly, erythropoietin (EPO), an endogenous glycoprotein, exerts multiple tissue protective effects by inhibiting ER stress and apoptosis. We investigated the role and potential mechanism of EPO on VC in chronic kidney disease (CKD) rats and cultured vascular smooth muscle cells (VSMCs). The calcification model was established by subtotal nephrectomy in vivo or phosphate overload in vitro. The protein level of EPO receptor (EPOR) was increased in the calcified aortas of CKD rats. EPO prevented the reduction of VSMC phenotypic markers, and reversed the increased calcium content and calcium salt deposition in the aortas of CKD rats and cultured calcified VSMCs. The protein levels of activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94) were upregulated in aortas and VSMCs under calcifying conditions, indicating ER stress activation. EPO treatment of CKD rats or calcified VSMCs downregulated the protein levels of ATF4 and GRP94. Furthermore, ER stress-mediated apoptosis, determined by the protein levels of CCAAT⁄enhancer-binding protein-homologous protein and cleaved caspase 12, was increased in tunicamycin or calcification media-treated VSMCs, but the increased effect was reversed in EPO-treated groups. The increased apoptotic cells in calcified VSMCs, as indicated by Hoechst staining and flow cytometry, were downregulated by the co-administration of EPO or 4-phenyl butyric acid. In conclusion, EPO might attenuate VC by inhibiting ER stress mediated apoptosis through EPOR signaling.

Transmembrane Protein Aptamer Induces Cooperative Signaling by the EPO Receptor and the Cytokine Receptor β-Common Subunit.

SummaryThe erythropoietin receptor (EPOR) plays an essential role in erythropoiesis and other cellular processes by forming distinct signaling complexes composed of EPOR homodimers or hetero-oligomers between the EPOR and another receptor, but the mechanism of heteroreceptor assembly and signaling is poorly understood. We report here a 46-residue, artificial transmembrane protein aptamer, designated ELI-3, that binds and activates the EPOR and induces growth factor independence in murine BaF3 cells expressing the EPOR. ELI-3 requires the transmembrane domain and JAK2-binding sites of the EPOR for activity, but not the cytoplasmic tyrosines that mediate canonical EPOR signaling. Instead, ELI-3-induced proliferation and activation of JAK/STAT signaling requires the transmembrane and cytoplasmic domains of the cytokine receptor β-common subunit (βcR) in addition to the EPOR. Moreover, ELI-3 fails to induce erythroid differentiation of primary human hematopoietic progenitor cells but inhibits nonhematopoietic cell death induced by serum withdrawal.

Deletion of a flippase subunit Tmem30a in hematopoietic cells impairs mouse fetal liver erythropoiesis.

Transmembrane protein 30A (Tmem30a) is the β-subunit of P4-ATPases which function as flippase that transports aminophospholipids such as phosphatidylserine from the outer to the inner leaflets of the plasma membrane to maintain asymmetric distribution of phospholipids. It has been documented that deficiency of Tmem30a led to exposure of phosphatidylserine. However, the role of Tmem30a in vivo remains largely unknown. Here we found that Vav-Cre-driven conditional deletion of Tmem30a in hematopoietic cells led to embryonic lethality due to severe anemia by embryonic day 16.5. The numbers of erythroid colonies and erythroid cells were decreased in the Tmem30a deficient fetal liver. This was accompanied by increased apoptosis of erythroid cells. Confocal microscopy analysis revealed an increase of localization of erythropoietin receptor to areas of membrane raft microdomains in response to erythropoietin stimulation in Ter119−erythroid progenitors, which was impaired in Tmem30a deficient cells. Moreover, erythropoietin receptor (EPOR)-mediated activation of the STAT5 pathway was significantly reduced in Tmem30a deficient fetal liver cells. Consistently, knockdown of TMEM30A in human CD34+ cells also impaired erythropoiesis. Our findings demonstrate that Tmem30a plays a critical role in erythropoiesis by regulating the EPOR signaling pathway through the formation of membrane rafts in erythroid cells.