Mature Red Cells Research Articles

Oxidation and erythropoiesis.

Erythropoiesis is a complex multistep process going from committed erythroid progenitors to mature red cells. Although recent advances allow the characterization of some components of erythropoiesis, much still remains to be investigated particularly on stress erythropoiesis. This review summarizes recent progresses made to understand the impact of oxidative stress on normal and pathologic erythropoiesis. During erythroid maturation, reactive oxygen species might function as second messenger through either transient oxidation of cysteine residues on signaling targets or modulation of intracellular signaling pathways. Thus, in erythropoiesis, efficient cytoprotective systems are required to limit possible reactive oxygen species-related toxic effects especially in stress erythropoiesis characterized by severe oxidation such as β-thalassemia. In addition, prolonged or severe oxidative stress impairs autophagy, which might contribute to the block of erythroid maturation in stress erythropoiesis. Understanding the functional role of cytoprotective systems such as peroxiredoxin-2 or classical molecular chaperones such as the heat shock proteins will contribute to develop innovative therapeutic strategies for ineffective erythropoiesis. We provide an update on cytoprotective mechanisms against oxidation in normal and stress erythropoiesis. We discuss the role of oxidative sensors involved in modulation of intracellular signaling during erythroid maturation process in normal and stress erythropoiesis.

Improvement of Red Blood Cell Maturation In Vitro by Serum-Free Medium Optimization.

Despite recent studies on media additives, the low viability and dysplastic features of terminally mature erythroid cells are still major problems in enhancing erythroid cell yields in vitro. Moreover, research on enhancing terminal erythropoiesis has been focused on the immature stage of erythroid cells such as burst forming unit-erythroid and colony forming unit-erythroid. Here, we tested many commercially available serum-free culture media and developed a superior culture media formulation compared with the conventional control for higher expansion-fold, higher viability, and therefore enhanced red cell productivity. The addition of the specific medium to the previously known best media at a specific ratio, whose effects were not dose-dependent, enabled the generation of significantly higher erythrocyte products with over 1.3 million-fold proliferation of erythroid cells after maintenance for 21 days throughout the maturation stages from CD34+ cells. The cells cultured in this condition expressed maturation markers and were significantly superior in differentiation and enucleation. Comparative mRNA profiling revealed that erythroid cells in this medium showed more efficient maturation in mRNA levels. The cultured cells showed comparable erythroblast survival and also restored better red blood cell (RBC) functions of oxygen saturation profile with expression of adult globin up to 99%. However, to develop chemically defined media, the well-known supplements including hormones, cytokines, and serum-replacement reagents were not sufficient to replace the optimized media in producing mature RBCs. Taken together, our optimized medium formulation under serum-free culture conditions produced the best reproducible results on productivity and maturation in erythroid cells with economic benefits. These culture conditions may thus serve as a useful platform for further investigation of in vitro erythropoiesis and to develop defined serum-free media for clinical trials.

In Patients with Minor Beta-Thalassemia, Cognitive Performance Is Related to Length of Education, But not to Minor Beta-Thalassemia or Hemoglobin Levels

Objective: Thalassemia is one of the most frequent monogenic disorders, leading to impairment in the maturation and survival of red blood cells. The question examined here is whether, and if so, to what extent, people with beta-thalassemia might also be impaired in their cognitive functioning. Previous results in adults with beta-thalassemia showed cognitive impairment when compared to healthy controls. However, length of education was never taken into consideration as a possible confounder. Accordingly, the aim of the present study was to assess people with minor beta-thalassemia and compare them to healthy controls, while controlling for length of education. Method : A total of 25 adults (mean age: 29.36 years; 56% females) with beta-thalassemia and 25 healthy controls (mean age: 27.84 years; 72% females) took part in this cross-sectional study. They underwent cognitive testing (executive functions, attention, working memory), and their haemoglobin levels were assessed. Results: Cognitive performance did not significantly differ between patients with minor beta-thalassemia and healthy controls. Irrespective of group, higher cognitive performance was strongly associated with time spent in education. No gender differences were observed. Conclusion: Compared to healthy controls, cognitive performance was not impaired among patients with minor beta-thalassemia when length of education was introduced as a further co-variate. In both patients with minor beta-thalassemia and healthy controls, higher cognitive performance was associated with time spent for education. Health professionals should inform patients with minor beta-thalassemia that cognitive performance is related to the length of education and not to the health status of minor beta-thalassemia per se.

Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation

AbstractErythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell's surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

Reduction of hemoglobin, not iron, inhibited maturation of red blood cells in male rats exposed to high intensity endurance exercises

The existence of sports anemia, induced by strenuous or long-term exercise and characterized by decreases of red blood cells (RBCs), hemoglobin and iron content, remains to be doubtful. To observe the effects of endurance exercise on RBCs and explain the underlying reason, we designed this study by observing RBCs parameters and iron metabolism in 8-weeks training rats and effects of iron supplement or protein supplement on RBCs. Results showed that erythrocyte counts, hematocrit, mean corpuscular volume and hemoglobin content decreased while RBC distribution width increased in exercised rats at later stage during 8 weeks training. But the contents of serum iron and ferritin decreased only at 1-week and 2-week and returned to normal at 4-week and 8-week. Same as iron content, apparent iron absorption rate was declined at early stage but restored to normal level at 8-week, as well as serum adrenaline, cortisol and insulin levels. Instead, the contents of total protein and albumin in serum were decreased at later stage during 8-weeks training. Furthermore, we observed that protein supplement ameliorated RBCs parameters in rats exposed to 8 weeks swimming exercise, but iron supplement had no effects on RBCs, though it obviously increased iron content of serum and the liver. Based on these results, we drew a conclusion that transient changes of iron metabolism, which may be induced by stress hormone changes, was not the reason for RBCs decrease in endurance exercises but hemoglobin reduction, induced by defects in protein supplement, impeded development of RBCs.

Autophagy in Mitochondrial Quality Control.

Autophagy plays an important role in the renewal of cellular components, which function in energy production, metabolism, and clearance of damaged organelles. Both macroautophagy and microautophagy are involved in these processes. Although it was thought that nonselective macroautophagy is responsible for the clearance of damaged or old organelles, recent studies show that the clearance of cellular organelles depends on selective processes. Mitophagy is a process for selective degradation of mitochondria, which is well documented. The selective autophagy for other organelles includes endoplasmic reticulum autophagy (reticulophagy) and peroxisome autophagy (pexophagy). Autophagy is a routine pathway for cells to degrade unused proteins and damaged organelles in cells. Autophagy selectively removes dysfunctional cellular components but not damages the normally functioning organelles, to maintain the homeostasis of cells. In addition to the maintenance of the homeostasis of cells, autophagy clears the damaged organelles in disease or injury conditions to achieve cellular quality control. In some differentiated cells, such as red blood cells, some organelles are removed during the maturation, including mitochondria. The autophagy system can selectively clear the mitochondria and other organelles, which lead to the maturation of red blood cells. Dysfunction of autophagy impairs the clearance of damaged organelles, which results in injury of cells. In the maturation of red blood cells, failure to clear the cellular organelles by autophagy will disturb the normal differentiation of red blood cells, leading to a series of diseases such as anemia.

Global Chromatin Occupancy and Epigenetic Signature Analysis Reveal New Insights into the Function of GATA1 N-Terminus in Erythropoiesis

Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. DisclosuresCrispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Optimized Beta-Globin Expression and Enucleation from Induced Red Blood Cells for In Vitro Modeling of Sickle Cell Disease

Human induced pluripotent stem cells (hiPSCs) hold remarkable capacity for disease modeling and the development of novel therapeutic treatments for sickle cell disease (SCD). hiPSCs can theoretically produce all cell types including induced red blood cells (iRBCs). Sickle cell patients, in particular, could benefit from autologous, engineered red blood cells (RBCs). Many patients possess rare Rh phenotypes, are allo-sensitized to blood products and are at risk of iron overload from recurrent transfusions. Therefore, the generation of personalized iRBCs is attractive. Yet, in vitro iRBC production has been hampered by an inability of these cells to differentiate into terminally-mature, enucleated, beta globin-expressing RBCs. Here, we describe updated strategies to improve in vitro production of iRBCs. hiPSCs from sickle cell patients as well as those with normal hemoglobin were differentiated into hematopoietic stem progenitor cells (HSPCs) and immortalized via the overexpression of a previously characterized set of transcription factors promoting self-renewal and multipotency under the control of a doxycycline-regulated promoter. Utilizing an in vitro protocol incorporating increasing concentrations of human plasma, HSPCs differentiated from these lines proceed through terminal erythroid differentiation, including the formation of CD71-/GlyA+/α4 integrin-/Band 3+ cells. Plasma-stimulated iRBCs achieved robust enucleation (11-60.7%) and underwent fetal to adult globin-switching. Further, nearly 21% of the enucleated iRBCs were RNA negative erythrocytes 5-8 microns in diameter. RNA sequencing analysis reveals similar transcriptional profiles between iRBCs and peripheral blood CD34+- derived cultured RBCs (cRBCs) yet distinct differences between SCD and WT iRBCs. SCA iRBCs have increased extracellular matrix organization, cell-cell adhesive properties and up-regulation of hypoxia-response genes. Heme metabolism, DNA repair, fatty acid metabolism and oxidative phosphorylation are all impaired in SCD iRBCs. Assessment of cell physiology exposes membrane damage in SCD iRBCs with increased phalloidin permeability in comparison to wild type controls. Intriguingly, SCD iRBCs co-expressing gamma and beta-globin also demonstrate sickling under hypoxic conditions. With the development of expandable source of erythroid progenitors capable of producing mature red cells, we now aim to utilize this platform for robust disease modeling and autologous cell therapy. [Display omitted] DisclosuresHeeney:Pfizer: Research Funding; Sancilio Pharmaceuticals: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee.

Heme-Regulated Inhibitor (HRI) Loss and Pharmacologic Treatments Cooperate to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF for SCD patients but is limited in its efficacy. Recent work in our laboratory utilizing a protein kinase-domain focused CRISPR-Cas9 based genetic screen identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 361:6399, 285-290). Depleting HRI in a human erythroid cell line and primary human erythroid cultures significantly raised HbF levels. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. While all these approaches induce HbF in vitro, it remains to be seen whether these would achieve sufficient levels of HbF induction in patients when used as single agents. Although the specific mechanisms by which HRI, HU, pomalidomide and UNC0638 regulate HbF remain to be fully resolved, we hypothesized that they might work in distinct pathways, and that by combining them might improve effect size and diminish potential adverse effects on erythroid cell maturation and viability.We utilized a three-stage in vitro culture system that recapitulates normal erythropoiesis by terminal differentiation of adult CD34+ hematopoietic cells. Primary human CD34+ cells were treated with select pharmacologic inducers of HbF, including hydroxyurea, pomalidomide, or UNC0638, in combination with shRNA lentiviral knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and cation-exchange HPLC. We find that knockdown of HRI leads to significant HbF induction as expected. We further observed an inverse correlation between remaining HRI levels and HbF induction with maximal effect size requiring 85%-90% HRI depletion. HU displayed comparatively little activity and failed to increase the effects of HRI knockdown. However, treatment with pharmacologic levels of pomalidomide or UNC0638 combined with HRI depletion each showed greater than additive effects in HbF with levels reaching 25-30% HbF for UNC0638-treated and 30-40% HbF for pomalidomide-treated combinations, suggesting underlying cooperativity of HbF induction. RT-qPCR and Western blot analyses suggest that the diminished expression of the HbF repressor BCL11A accounts in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which Bcl11A depletion was greater than 90%, while other HbF repressors such as LRF were unchanged. To determine whether HbF induction results in reduced sickle cell formation, we performed combination HRI depletion and HbF pharmacologic induction in primary CD34+ cells derived from sickle cell patients. HRI depletion in primary SCD cells showed a marked increase in HbF from baseline levels; in addition, significant cooperativity with pomalidomide and UNC0638 was observed, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations with no apparent detrimental effects on erythroid differentiation or maturation. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype in vitro.Overall, we find that the combination of HbF pharmacologic induction and shRNA-mediated HRI inhibition results in significant cooperative upregulation of HbF levels in both normal and sickle cell-derived primary human cells without impairing red cell maturation. We are currently exploring additional potential synergies with HbF regulators to determine the optimal modalities to maximize HbF induction. While no effective and specific HRI inhibitors are currently available, our work suggests that future small molecule inhibition of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, β, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1−/−, Ampk β1−/− and Ampk γ1−/− mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis

AbstractDuring erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼109 molecules of heme must be synthesized, consuming 1010 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle. Based upon the observation that 1 subunit of succinyl-CoA synthetase (SCS) physically interacts with the first enzyme of heme synthesis (5-aminolevulinate synthase 2, ALAS2) in erythroid cells, it has been posited that succinyl-CoA for ALA synthesis is provided by the adenosine triphosphate–dependent reverse SCS reaction. We have now demonstrated that this is not the manner by which developing erythroid cells provide succinyl-CoA for ALA synthesis. Instead, during late stages of erythropoiesis, cellular metabolism is remodeled so that glutamine is the precursor for ALA following deamination to α-ketoglutarate and conversion to succinyl-CoA by α-ketoglutarate dehydrogenase (KDH) without equilibration or passage through the TCA cycle. This may be facilitated by a direct interaction between ALAS2 and KDH. Succinate is not an effective precursor for heme, indicating that the SCS reverse reaction does not play a role in providing succinyl-CoA for heme synthesis. Inhibition of succinate dehydrogenase by itaconate, which has been shown in macrophages to dramatically increase the concentration of intracellular succinate, does not stimulate heme synthesis as might be anticipated, but actually inhibits hemoglobinization during late erythropoiesis.

Induced pluripotent stem cell–based mapping of β-globin expression throughout human erythropoietic development

AbstractRobust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish β-globin–expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with β-globin–expressing cells showing increased maturation. Compared with other cells, β-globin–expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.

Increased Reactive Oxygen Species and Cell Cycle Defects Contribute to Anemia in the RASA3 Mutant Mouse Model scat.

RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a missense mutation in Rasa3. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in scat is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in scat and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In scat mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G0/G1 phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G1 checkpoint. However, this increase in G1 is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in scat; p = 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the scat bone marrow at the proerythroblastic (15.3%; p = 0.004), polychromatophilic (8.5%; p = 0.01), and orthochromatic (4.2%; p = 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in scat. Finally, the cell cycle defect and increased levels of ROS suggest that scat is a model of bone marrow failure with characteristics of aplastic anemia.

Sickle cell dehydration: Pathophysiology and therapeutic applications.

Cell dehydration is a distinguishing characteristic of sickle cell disease and an important contributor to disease pathophysiology. Due to the unique dependence of Hb S polymerization on cellular Hb S concentration, cell dehydration promotes polymerization and sickling. In double heterozygosis for Hb S and C (SC disease) dehydration is the determining factor in disease pathophysiology. Three major ion transport pathways are involved in sickle cell dehydration: the K-Cl cotransport (KCC), the Gardos channel (KCNN4) and Psickle, the polymerization induced membrane permeability, most likely mediated by the mechano-sensitive ion channel PIEZO1. Each of these pathways exhibit unique characteristics in regulation by oxygen tension, intracellular and extracellular environment, and functional expression in reticulocytes and mature red cells. The unique dependence of K-Cl cotransport on intracellular Mg and the abnormal reduction of erythrocyte Mg content in SS and SC cells had led to clinical studies assessing the effect of oral Mg supplementation. Inhibition of Gardos channel by clotrimazole and senicapoc has led to Phase 1,2,3 trials in patients with sickle cell disease. While none of these studies has resulted in the approval of a novel therapy for SS disease, they have highlighted the key role played by these pathways in disease pathophysiology.

Effects of Electromagnetic Fields Modulated by Infralow Frequencies on the Production of Stem Cells

Experimental validation of the method for regulation of stem cell proliferation and differentiation is carried out. The method consists in exposure to ultrahigh frequency electromagnetic radiation, modulated by infralow frequencies with variable parameters. A specially designed programmer, setting up the parameters of exposure, is connected to the source of radiation. The zones of anatomical location of the red bone marrow of rats were exposed for 15 min to the amplitude-modulated electromagnetic radiation of ultrahigh frequency range. The parameters of exposure were determined in previous studies. The red bone marrow was collected from the sternum and head of the femur. The cellular composition of the red bone marrow was evaluated 1, 2, 3, and 6 days after the exposure. The optimal therapeutic mode of irradiation was then chosen, leading to stem cell activation with subsequent proliferation and differentiation into mature red bone marrow cells.

Examining the CREB Transcriptional Program in Erythropoiesis

The erythropoietin (EPO) signal transduction network regulates a plethora of critical cellular functions during red blood cell maturation including proliferation and iron metabolism. Recently, it has been discovered that EPO signaling also controls heme metabolism through activation of the protein kinase A (PKA) pathway. The activation of PKA by EPO leads to the mitochondrial phosphorylation of the terminal heme enzyme, ferrochelatase (FECH), to ramp up heme production. In addition to FECH, a second principal PKA target is the CREB transcription factor, leading to the hypothesis that the EPO-PKA signaling pathway also regulates gene expression during red cell maturation. Here, we tested this hypothesis using biochemical and bioinformatics approaches. We found that CREB becomes phosphorylated in human and murine erythroid cells in an EPO-dependent manner. Using pharmacologic inhibitors, we confirmed that CREB phosphorylation is downstream of JAK2 and PKA signaling. Moreover, we found enrichment of a previously identified CREB-gene signature within the erythroid transcriptional program. To date, our work suggests that CREB plays a role during red cell development as an effector of EPO signaling. Further work will be required to understand the function of CREB target genes. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic-Specific CSNK2B Loss in Mice Causes Impaired Erythropoiesis

CK2 (Csnk2, casein kinase 2) is a Ser-Thr kinase composed by two catalytic (α) and two regulatory (β) subunits and involved in the regulation of various signaling cascades, which are critical for stem cell biology and hematopoietic development. However, a direct role for CK2 during blood cell differentiation is still undefined. Here, we examined the function of CK2 in erythropoiesis by using a hematopoietic-specific conditional knockout mouse model of the β regulatory subunit (Vav1-CRE x Csnk2β f/f mice). Since CK2β knockout mice died in utero, the study was carried out during gestation collecting fetuses from 12.5 to 17.5 days post conception (dpc) and performing the analysis on fetal liver. CK2β knockout fetuses were pale and hydropic, displayed a smaller liver, disarrayed vascularization and haemorrhages. Lack of CK2β caused depletion of hematopoietic/precursor cells, in particular of common lymphoid progenitors and megakaryocyte-erythrocyte progenitors. CK2β loss resulted to affect both early and late erythroid maturation and red cell viability. CK2β knockout contained lower numbers of TER119 positive cells, which displayed a down modulation of the surface expression of transferrin receptor (CD71) and an increased spontaneous apoptosis. Erythroid cells showed alterations in morphology compatible with myelodysplastic changes. Loss of CK2β caused alterations of erythroid cell proliferation, which was different depending on the stage of erythroid maturation: indeed, BrdU and 7AAD staining showed that less mature erythroid cells (CD71+Ter119-) had a lower rate of proliferation but a normal viability; on the contrary, more mature (CD71-Ter119+) erythroid cells suffered in part of apoptosis and in part accumulated in the S phase. RNA seq analysis performed on purified Ter119+ cells revealed upregulation of TP53 -associated genes as well as of Cdkn1a (p21); on the contrary, there was a down-modulation of Stat5 (an erythropoietin receptor down-stream effector) and genes involved in red cell survival and differentiation in particular c-kit and genes associated to the PI3/Akt pathway. The expression of adhesion molecules and surface carriers for inorganic cations/anionsimportant for the osmotic equilibrium and cell membrane integrity was also found markedly dysregulated. Real time quantitative PCR and Western Blot (WB) analyses confirmed the expression data of Cdkn1a, c-Kit, Bcl-xL, Jak-Stat5 as well as of Akt-Gata-1 axis. Gata-1, the key transcription factor for definitive erythropoiesis, was reduced in CK2β knockout mice as were its downstream target genes such as Alas-2, Lrf, Eklf, Epo-R, β-globin. Immature fetal globins accumulated. In order to find a molecular mechanism, we used an in vitro model of erythroid differentiation based on G1ER cells, an estrogen inducible GATA-1 null murine erythroblast cell line; the combined treatment of β-estradiol and inhibition of CK2 through the chemical inhibitor CX-4945 or RNA interference against CK2β confirmed the negative effect on differentiation. Western blot analysis indicated a potential role of the kinase in the regulation of Akt, Gata-1 and Stat5 protein stability. Moreover, the blockade or down modulation of CK2 caused changes in Gata-1 nuclear distribution with loss of the speckled pattern induced by β-estradiol. Thus, CK2 is a likely essential controller of GATA-1 transcriptional function. Altogether, our work demonstrates that CK2 is a master regulator of erythroid development, by impinging on Stat5, Akt and Gata-1 signaling and influencing red cell viability, bioenergetics, proliferation and maturation. DisclosuresNo relevant conflicts of interest to declare.

Fyn Kinase Is Involved in EPO Receptor Signaling and Is Required to Harmonize the Response to Oxidation

Erythropoiesis is a complex multistep process during which committed erythroid progenitors undergo terminal differentiation to produce circulating mature red cells. Erythroid differentiation is characterized by the production of reactive oxygen species (ROS) both in response to erythropoietin (EPO) and to the large amount of iron imported into the cells for heme biosynthesis. During erythropoiesis, ROS might function as second messenger by modulating intracellular signaling pathways. Fyn, an Src kinase, has been previously reported to participate in signaling pathways in response to ROS in various cell types. Here, we explore the potential contribution of Fyn to normal and stress erythropoiesis by studying 2-4 months-old Fyn knockout mouse strain (Fyn-/-) and C57B6/2J as wild-type controls. Fyn-/- mice showed a mild compensated microcytic anemia associated with signs of dyserythropoiesis. Increased ROS levels and Annexin-V+ cells were presented in all Fyn-/- erythroblast subpopulations compared to wild-type, suggesting a possible reduction in the efficiency of erythropoietin (EPO) signaling pathway in the absence of Fyn. Indeed, in Fyn-/- erythroblasts we observed a reduction in Tyr-phosphorylation state of EPO-R associated with a compensatory activation of Jak2 without major change in Lyn activity. A reduction in STAT5 activation resulting in down-regulation of Cish, a known direct STAT5 target gene, was noted in Fyn-/- erythroblasts. This was paralleled by a reduction in GATA1 and increased HSP70 nuclear translocation compared to wild type, supporting a higher cellular pro-oxidant environment in the absence of Fyn. Using the vitro cell forming colony unit assay, we found a lower in CFU-E and BFU-E cells production, which once again was associated with decreased activation of EPO mediated cascade in the absence of Fyn. To explore the possible role of Fyn in stress erythropoiesis, mice were treated with either phenylhydrazine (PHZ) or doxorubicin (Doxo). Fyn-/- mice showed prolonged anemia after either PHZ or Doxo treatment with a delayed hematologic recovery compared to wild-type animals. When we analyzed the expression of a battery of ARE-genes related to oxidative response such as catalase, Gpx, heme-oxygenase 1 and peroxiredoxin-2, we noted up-regulated expression of these genes in sorted Fyn-/- erythroblasts compared to wild-type cells. In agreement, we observed increased activation of the redox-sensitive transcriptional factor Nrf2 targeting ARE-genes, whose regulation has been previously linked to Fyn. In fact, Nrf2 is switched-off by Fyn, ubiquitylated and delivered to the autophagosome by the p62 cargo protein. In Fyn-/- sorted erythroblasts, we observed (i) accumulation of p62 in large clusters; and (ii) reduction of Nrf2-p62 complex compared to wild-type cells. To address the question whether the perturbation of Nrf2-p62 system results in impairment of autophagy in the absence of Fyn, we used Lysotrack to explore late phases of autophagy. Lysosomal progression was defective in Fyn-/- reticulocytes and it was associated with accumulation of p62 during in vitro reticulocyte maturation. These data indicate that the absence of Fyn blocks the Nrf2 post-induction response to oxidation, resulting in impaired autophagy. To validate our working hypothesis, we treated Fyn-/- mice with Rapamycin, an inducer of autophagy. In Fyn-/- mice, Rapamycin treatment resulted in decrease dyserythropoiesis, ROS levels and Annexin V+ cells, associated with reduction in accumulation of p62 in Fyn-/- erythroblasts. As a proof of concept, we treated both mouse strains with PHZ with or without Rapamycin. This latter worsened PHZ induced acute anemia in wild-type mice but not in Fyn-/- animals. Collectively, our data enabled us to document a novel role for Fyn in erythropoiesis, contributing to EPO-R activation and harmonizing the Nrf2-p62 adaptive cellular response against oxidation during normal and more importantly in stress erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

RASA3 Deficiency Contributes to Anemia By Multiple Mechanisms

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage.The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p<0.05).Studies exploring the influence of the galectin family on erythropoiesis and Ras signaling in the context of scat are underway.Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis.Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. DisclosuresNo relevant conflicts of interest to declare.

Unravelling pathways downstream Sox6 induction in K562 erythroid cells by proteomic analysis

The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6−/− red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562 cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation. To obtain an overview on processes downstream to Sox6 expression, we performed a differential proteomic analysis on human erythroid K562 cells overexpressing Sox6. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24 A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these proteins during erythropoiesis.