Role In Erythroid Maturation Research Articles

YAP and TAZ play a crucial role in human erythrocyte maturation and enucleation

BackgroundYes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) are two key transcription co-activators of the Hippo pathway. Both were originally characterized as organ size and cell proliferation regulators. Later studies demonstrated that the Hippo pathway may play a role in Drosophila and mammal hematopoiesis. However, the role of the Hippo pathway in human erythropoiesis has not yet been fully elucidated.MethodsThe role of YAP and TAZ was studied in human erythropoiesis and hematopoietic stem cell (HSC) lineage determination by using mobilized peripheral blood (PB) and cord blood (CB)-derived HSC as a model. HSCs were isolated and cultured in an erythroid differentiation medium for erythroid differentiation and culture in methylcellulose assay for HSC lineage determination study.ResultsYAP and TAZ were barely detectable in human HSCs, but became highly expressed in pro-erythroblasts and erythroblasts. Depletion or knockdown of YAP and/or TAZ did not affect the ability of HSC lineage specification to erythroid lineage in either methylcellulose assay or liquid culture. However, depletion of YAP and TAZ did impair erythroblast terminal differentiation to erythrocytes and their enucleation. Moreover, ectopic expression of YAP and TAZ in pro-erythroblasts did not exert an apparent effect on erythroid differentiation, expansion, or morphology.ConclusionsThis study demonstrated that YAP/TAZ plays important role in erythroid maturation and enucleation but is dispensable for lineage determination of human HSCs.

Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major.

Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major.

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche.

The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.

LSD1 Influences Both Histone and DNA Methylation During Terminal Erythroid Maturation

The molecular mechanisms that drive the maturation of a committed erythroid progenitor to a functional red blood cell are incompletely understood. LSD1 (Lysine-Specific Histone Demethylase 1) is a widely expressed histone demethylase that plays an important role in erythroid maturation (Kereyni, elife, 2013). Although LSD1 is important for a number of biologic processes ranging from embryonic development to leukemogenesis, the molecular mechanisms underlying the influence of LSD1 on gene expression are incompletely understood. The goal of our study is to elucidate the molecular mechanisms by which LSD1 regulates erythroid gene expression and influences erythroid maturation. We hypothesize that LSD1 promotes specific patterns of histone and DNA methylation that facilitate gene expression changes necessary for normal erythroid maturation to occur. To address this hypothesis, the functional and molecular consequences of LSD1 knockdown were assessed in Extensively Self Renewing Erythroblasts (ESREs), a non-transformed, karyotypically normal model of terminal erythroid maturation (England, Blood, 2011). Primary fetal liver was cultured in the presence of EPO, SCF, IGF1 and dexamethasone to derive ESREs. The ESREs were capable of extensive ex-vivo expansion, doubling daily at the proerythroblast phase, however when matured, >90% of cells became benzidine positive and >65% enucleated within 3 days. Lentiviral-mediated shRNA was used to knock down LSD1 in expanding ESREs. Imaging flow cytometry done on maturation day 3 demonstrated that the knockdown cells had impairments in multiple facets of maturation, with larger cell and nuclear areas, higher kit expression, and lower rates of enucleation than the scramble control. LSD1 knockdown was also associated with impaired hemoglobin accumulation (78% vs. 95% benzidine positive; p<0.005). Treatment of ESREs with an inhibitor to LSD1 (Tranylcypromine; TCP) resulted in similar abnormalities in cell and nuclear size, kit expression, hemoglobin accumulation, and enucleation (40% vehicle vs.1% TCP). The functional deficits in maturation, including abnormal kit expression and low rates of enucleation, persisted on maturation day 4.To delineate the molecular mechanisms underlying this maturation impairment, RNA-seq was done in LSD1 knockdown and scramble control samples, and 230 differentially expressed genes (FDR<0.01) were identified using cuffdiff (Trapnell, Nat Biotech, 2013). Consistent with LSD1's role in erythroid maturation, Ingenuity Pathway Analysis identified multiple networks involving hemoglobin synthesis, and GATA1, EPO, and KLF1 were all predicted as upstream regulators (p-values of 8.24e10-11, 7.25 e10-6, and 3.86e10-4, respectively). To better understand how LSD1 influences gene expression, chromatin immunoprecipitation coupled with high throughput sequencing was used to identify sites of H3K4me2 binding in the differentially expressed genes. 214/230 differentially expressed genes were associated with sites of H3K4me2 occupancy. Quantitative ChIP demonstrated that LSD1 inhibition was associated with increases in H3K4me2 levels at a subset of these sites, however consistent with previous studies, global levels of H3K4me2, determined by Enzyme Linked Immunosorbent Assay (ELIZA), did not change significantly.Although it is known that LSD1 demethylates and stabilizes the maintenance DNA methyltransferase DNMT1 (Wang, Nat Genet 2009), the consequences of LSD1 loss on DNA methylation (5-methyl cytosine; 5-mC) have yet to be investigated. To gain a comprehensive understanding of how LSD1 regulates erythroid gene expression, changes in the level of 5-mC were assessed after knockdown or inhibition of LSD1. Global 5-mC levels, determined by ELIZA assay, were ∼30% lower in TCP treated samples than vehicle treated control (p<0.02) and western blot demonstrated a 3-fold decrease in DNMT1 protein in the TCP treated samples. Both methyl binding domain pull-down coupled with quantitative PCR and genome-wide bisulfite sequencing were utilized to assess changes in 5-mC levels in the differentially expressed genes. Loss of LSD1 was associated with significantly lower levels of 5-mC at several differentially expressed, erythroid-specific genes, such as bh1. Taken together, these data support the hypothesis that LSD1 influences both histone and DNA methylation at genes important for erythroid maturation. Disclosures:No relevant conflicts of interest to declare.

Carbon Monoxide Induced Erythroid Differentiation of K562 Cells Mimics the Central Macrophage Milieu in Erythroblastic Islands

Growing evidence supports the role of erythroblastic islands (EI) as microenvironmental niches within bone marrow (BM), where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb) is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule - CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage.Based on the above, the study hypothesized CO to have a role in erythroid maturation. Thus, the effect of CO gas as a potential erythroid differentiation inducer on the common model for erythroid progenitors, K562 cells, was explored. Cells were kept under oxygen lacking environment to mimic BM conditions. Nitrogen anaerobic atmosphere (N2A) served as control for CO atmosphere (COA). Under both atmospheres cells proliferation ceased: in N2A due to cell death, while in COA as a result of erythroid differentiation. Maturation was evaluated by increased glycophorin A expression and Hb concentration. Addition of 1%CO only to N2A, was adequate for maintaining cell viability. Yet, the average Hb concentration was low as compared to COA. This was validated to be the outcome of diversified maturation stages of the progenitor's population.In fact, the above scenario mimics the in vivo EI conditions, where at any given moment only a minute portion of the progenitors proceeds into terminal differentiation. Hence, this model might provide a basis for further molecular investigations of the EI structure/function relationship.

Distribution and shedding of the membrane phosphatidylserine during maturation and aging of erythroid cells

Maturation and aging of erythroid cells are accompanied by extensive remodeling of the membrane and a marked decrease in cell size, processes that are mediated by externalization and shedding of phosphatidylserine (PS). In the present study, we investigated the redistribution of PS in the plasma membrane of erythroid precursors during their maturation and of mature RBCs during senescence, and the involvement of changes in calcium (Ca)-flux in these processes. Maturation was studied by analyzing normal human bone marrow cells as well as cultured human normal erythroid precursors induced by erythropoietin and murine erythroleukemia cells induced by hexamethylene-bisacetamide. Senescence was studied in normal human peripheral RBCs following density fractionation. PS and Ca were determined by flow cytometry using annexin-V and Flu-3, respectively. The outer, inner and shed PS were quantified by a novel two-step binding inhibitory assay. The results indicate a bi-phasic modulation of intracellular Ca and PS externalization/shedding; both of which decreased during maturation and increased during aging. The role of intracellular Ca in PS externalization/shedding was demonstrated by modulating intracellular Ca: Ca was decreased by incubating the cells with an ion chelator (EDTA) or with decreasing concentrations of Ca, whereas treatment with the ionophore A23187 elevated intracellular Ca. The results showed that low Ca resulted in decreased outer and shed PS, whereas high Ca had the opposite effect. The results suggest that PS externalization and shedding are mediated by increased cellular Ca-flux, and that they play an important role in erythroid maturation and RBC senescence.

Identification of erythropoietin‐induced microRNAs in haematopoietic cells during erythroid differentiation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through mRNA degradation or translation inhibition. It has not yet been clearly elucidated whether miRNAs participate in haematopoietic processes such as cell differentiation, apoptosis and maintenance of adequate levels of circulating blood cells. A human miRNA microarray was used to analyze miRNA expression in the erythropoietin-dependent cell line UT-7/EPO compared to other factor-dependent UT-7 cell lines. Among 324 human miRNAs, MIRN188, MIRN362 and MIRN210 levels were significantly elevated in UT-7/EPO cells, and stimulation with EPO in UT-7 cells increased the level of these three miRNAs. Notably, knockdown of MIRN210 in UT-7/EPO cells led to apoptosis. In mouse fetal liver cells, MIRN210 expression was twofold higher in TER-119-positive cells than in TER-119-negative cells. The expression of MIRN210 was elevated during erythroid maturation in vitro. These data suggest MIRN210 to be a member a new class of regulatory miRNAs that might play an important role in erythroid maturation.

Study of Uncoupling Protein 2 (UCP2) Function during Erythroid Differentiation and Maturation.

Mitochondrial oxidative stress is thought to play a key role in sideroblastic anemia and the myelodysplastic syndrome. Potential sources of reactive radicals reside in the heme biosynthetic pathway involving the import and production of pro-oxidant agents, such as ALA and iron and in the respiratory chain. Antioxidant mechanisms are, therefore, expected to be an integral function in erythroid differentiation and their impairment is expected to affect hemoglobinization and maturation. The mitochondrial uncoupling proteins have been shown to reduce oxidative stress through the generation of proton leak across the inner membrane of the mitochondria. They have been implicated in a wide range of physiological and pathological states, including obesity, diabetes, aging neurodegenerative, and immunological diseases.Here we report that UCP2 is induced during erythroid differentiation and that UCP2 deficient mice have a delayed recovery from anemia.We hypothesized that erythroid heme biosynthesis is accompanied by oxidative stress, which results in the induction of UCP2, and that UCP2 plays a role in erythroid maturation by preventing oxidative stress and damage.We found that UCP2 transcripts and protein are induced following the activation of GATA-1 in G1ER cells and during DMSO, butyrate and heme -induced differentiation of murine erythroleukemic (MEL) and K652 erythroid cell lines. Similarly, differentiation of primary mouse c-kit+ / Ter119− erythroid progenitors to Ter119+ is accompanied by induction of UCP2 transcripts. To test the functional significance of UCP2 in erythroid differentiation we studied a UCP2 null mouse. Peripheral blood analysis from UCP2 KO mice revealed a mild elevation of the reticulocyte index as compared to wild type (WT C57BL/6J) mice, which may be related to mild anemia. To test the role of UCP2 in recovery from anemia, we treated WT and UCP2 KO mice with phenylhydrazine for 3 days and studied erythropoiesis using FACS analysis of Ter119 and CD71 surface markers in cells isolated from bone marrow. Stimulation of erythropoiesis was more rapid in the WT mice as compared to the UCP2 KO. The delay in the mutant is more pronounced at the stage of the proerythroblast and is also reflected in the peripheral blood where a higher level of reticulocytes was transiently observed. By 9 days the UCP2 KO mice peripheral blood count was identical to the WT. Analysis of oxidative damage confirmed that UCP2 acts to reduce oxidative damage of mitochondrial proteins. The delayed reticulocytosis could not however be explained by cell death or by reduced hemoglobinization. The increased oxidative damage present in the UCP2 null cells during erythroid differentiation and maturation did not result in the stimulation of apoptosis as revealed by identical Annexin V staining profile of UCP2 KO and WT mice. Remarkably, iron incorporation and hemoglobin content assays ruled out a function of UCP2 in the process of heme biosynthesis per-se. We therefore conclude that UCP2 deficiency regulates maturation in the erythroid lineage independent of the heme biosynthetic pathway.

Modulation of the expression and activity of cyclooxygenases in normal and accelerated erythropoiesis

The present study was aimed at characterizing the expression and activity of cyclooxygenase (COX) isoenzymes in erythropoiesis. The expression and activity of cyclooxygenase (COX) and prostaglandin (PG) synthases were investigated in: 1) erythroblasts developed in culture from human CD34(+) hematopoietic progenitors, 2) erythroblasts in bone marrow specimens, and 3) peripheral erythrocytes isolated from healthy donors and from patients with a high regeneration rate of erythrocytes. While COX-1 protein was observed at each stage of erythroblast development, COX-2 protein was induced at later stages through a p38/MAPK-dependent pathway. Both COX isoforms were also observed in mature erythroblasts of the bone marrow. Erythroblasts developed in culture synthesized significantly more PGE(2) than TXB(2) and indomethacin delayed erythroid maturation. COX-1 and COX-2 were also observed in erythrocytes by immunostainings, although COX expression was confined to a fraction of circulating erythrocytes. Peripheral erythrocytes synthesized low but detectable amounts of PGE(2) and TXB(2). Similarly to erythroblast progenitors, PGE(2) was the prevalent prostanoid released by erythrocytes. This biosynthetic capacity was significantly increased in erythrocytes from patients with accelerated erythropoiesis as compared to controls. Both COX isoforms are present and enzymatically active during human erythropoiesis, although with different kinetics, and COX-derived prostanoids may play a role in erythroid maturation. Furthermore, peripheral erythrocytes retain in part the capacity of expressing COX and synthesizing prostanoids, which may contribute to the hemostatic/thrombotic response to vascular injury in different diseases, including congenital hemolytic disorders.

Molecular Cloning and Characterization of a KRAB-Containing Zinc Finger Protein, ZNF317, and Its Isoforms

To identify zinc finger genes in human primary cultured erythroid progenitor cells, RT-PCR was performed using primers specific for the conserved sequence in zinc finger domains of mouse Friend of GATA-1. We identified a novel Krüppel-associated box (KRAB) zinc finger gene, ZNF317. Evaluation of full-length cDNA obtained by RACE showed that it encoded a protein composed of 13 Krüppel-like zinc fingers and a KRAB domain. RT-PCR analysis revealed four alternatively splicing products (ZNF317-1 through ZNF317-4). The ZNF317 gene has seven exons and is located in human chromosome 19p13. Northern analysis revealed that ZNF317-1 and ZNF317-2 transcripts were ubiquitously expressed, whereas ZNF317-3 and ZNF317-4 transcripts were detected only in lymphocytes, spleen, and lung. Competitive RT-PCR analysis showed that the expression of ZNF317 mRNA significantly decreased during erythroid maturation. In lymphocytes, ZNF317 expression was reduced in response to mitogenic stimulation. We propose that ZNF317 may play an important role in erythroid maturation and lymphoid proliferation.

Erythropoietin triggers a burst of GATA-1 in normal human erythroid cells differentiating in tissue culture.

GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation.