Wild-type Erythroblasts Research Articles

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia

AbstractX-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Epor Stimulates Rapid Cycling and Larger Red Cells during Mouse and Human Erythropoiesis

Erythroid terminal differentiation (ETD) entails cell divisions coupled to decreasing cell size. The tight link between the number of cell divisions and red cell size is apparent in nutritional deficiencies or genetic variants in which fewer cycles result in larger red cells. Here we investigated novel EpoR functions, finding that EpoR signaling disrupts the relationship between cell cycle number and cell size, simultaneously promoting rapid cycling and the formation of larger red cells.EpoR is essential for erythroblast survival, but it is unclear whether it has other non-redundant functions. To address this, we developed a genetic system in which we rescue mouse Epor -/- fetal liver progenitors from apoptosis by transduction with the anti-apoptotic protein Bcl-x L, and compare their ensuing differentiation with that of Epor -/- progenitors rescued with EpoR (Fig 1a). We found that the Bcl-x L survival signal, in the absence EpoR, supported formation of enucleated red cells. However, key ETD features were abnormal.First, Bcl-x L-transduced Epor -/- erythroblasts underwent slower and fewer cell cycles (Figure 1b), differentiating prematurely into enucleated red cells. Premature induction of the cyclin-dependent-kinase inhibitor p27 KIP1 was in part responsible for the fewer cycles in the absence of EpoR signaling. We confirmed that EpoR also stimulates rapid cycling in wild-type erythroblasts in vivo, using a mouse transgenic for a live-cell reporter of cell cycle speed.Second, using imaging flow cytometry, we found that Bcl-x L-transduced Epor -/- erythroblasts were smaller than EpoR-transduced Epor -/- cells (Fig 1c,d). By doubly transducing Epor -/- erythroblasts with both Bcl-x L and EpoR, we verified that EpoR absence, and not Bcl-x L overexpression, is responsible for the smaller size of Bcl-x L-transduced Epor -/- erythroblasts and reticulocytes.Bcl-x L-transduced Epor -/- erythroblasts failed to upregulate the transferrin receptor, suggesting that iron deficiency may be responsible for their smaller size. However, neither iron supplementation, nor transduction with the transferrin receptor, rescued their smaller size. Iron regulates cell size through Heme-regulated eIF2α kinase (HRI). To definitively test the role of iron and HRI, we generated mice doubly deleted for both EpoR and HRI. We then rescued both Epor -/- and Epor -/-Hri -/- -fetal liver cells in parallel, by transduction with either Bcl-x L or EpoR. In agreement with the known role of HRI as a negative regulator of erythroblast size, both Bcl-x L- transduced and EpoR-transduced erythroblasts were larger on the Epor -/-Hri -/- genetic background. However, the difference in size between Bcl-x L and EpoR-rescued erythroblasts persisted in Epor -/-Hri -/- erythroblasts and reticulocytes (Fig 1c,d), conclusively showing that EpoR signaling regulates cell size independently of the HRI pathway.EpoR promoted increased erythroblast and reticulocyte cell size in wild-type mice in vitro and in vivo, in response to Epo concentrations ranging from 10 to 10,000 mU/ml. We also evaluated the effect of Epo on red cell size in humans, in two independent studies, where healthy volunteers were administered Epo for either 3 weeks (20 IU /kg every 48 hours, 25 subjects, Study #1) or for 7 weeks (weekly Epo dosing that increased hemoglobin by 10 -15%; 24 subjects, Study #2). In a third intervention, 21 subjects participated in a randomized double-blind placebo-controlled crossover study in which 900 ml of whole blood was withdrawn from the treatment group by venipuncture. In all three studies, the increase in MCV in the treatment groups persisted long after Epo and reticulocyte levels returned to baseline (Figure 2). There was no correlation between MCV and the reticulocyte count, whose time courses were clearly divergent (r < 0.1, Pearson's product-moment correlation). Further, computational simulation suggests that the extent and duration of the increase in MCV is unlikely to be the result of skewing of the circulating red cell population in favor of younger, larger red cells.Our work reveals a paradoxical EpoR-driven increase in erythroblast cycling simultaneously with increased erythroblast and red cell size. It suggests that EpoR alters the relationship between cell cycle and biomass in erythroblasts. It further suggests that hypoxia, anemia and other high-Epo syndromes are new diagnostic interpretations of increased MCV in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of South East Asian Ovalocytic Band 3 Disrupts Erythroblast Cytokinesis and Reticulocyte Maturation.

Southeast Asian Ovalocytosis results from a heterozygous deletion of 9 amino acids in the erythrocyte anion exchange protein AE1 (band 3). The report of the first successful birth of an individual homozygous for this mutation showed an association with severe dyserythropoietic anemia. Imaging of the proband’s erythrocytes revealed the presence of band 3 at their surface, a reduction in Wr(b) antigen expression, and increases in glycophorin C, CD44, and CD147 immunoreactivity. Immunoblotting of membranes from heterozygous Southeast Asian Ovalocytosis red cells showed a quantitative increase in CD44, CD147, and calreticulin suggesting a defect in reticulocyte maturation, as well as an increase in phosphorylation at residue Tyr359 of band 3, and peroxiredoxin-2 at the membrane, suggesting altered band 3 trafficking and oxidative stress, respectively. In vitro culture of homozygous and heterozygous Southeast Asian Ovalocytosis erythroid progenitor cells produced bi- and multi-nucleated cells. Enucleation was severely impaired in the homozygous cells and reduced in the heterozygous cells. Large internal vesicular accumulations of band 3 formed, which co-localized with other plasma membrane proteins and with the autophagosome marker, LC3, but not with ER, Golgi or recycling endosome markers. Immunoprecipitation of band 3 from erythroblast cell lysates at the orthochromatic stage showed increased interaction of the mutant band 3 with heat shock proteins, ubiquitin and cytoskeleton proteins, ankyrin, spectrin and actin. We also found that the mutant band 3 forms a strong interaction with non-muscle myosins IIA and IIB, while this interaction could not be detected in wild type erythroblasts. Consistent with this, the localization of non-muscle myosin IIA and actin was perturbed in some Southeast Asian Ovalocytosis erythroblasts. These findings provide new insights toward understanding in vivo dyserythropoiesis caused by the expression of mutant membrane proteins.

3132 - Modeling Defective Erythropoiesis in Myelodysplastic Syndrome (MDS)

Anemia is a significant clinical component of MDS, a clonal disorder predisposing to the development of leukemia. A murine model of MDS based on genetic defects in Nup98-HOXD13 (NHD13) recapitulates the peripheral cytopenias seen in patients, including macrocytic anemia (Balderman, 2016). We found that anemia in NHD13 mice was associated with a significant decrease in erythroid-specific progenitors (BFU-E), a lack of erythroblast precursor expansion, and decreased numbers of erythroblastic islands. To investigate whether microenvironmental defects contribute to development of anemia, we analyzed erythropoiesis in chimeric mice transplanted with UBC-GFP+ wild-type marrow and unlabeled cells from either NHD13 or wild-type mice. Non-anemic NHD13 chimeric mice developed macrocytosis. Surprisingly, almost all circulating red cells were GFP+, indicating their derivation from wild-type erythroblasts. In the marrow of these mice, we found significant decreases in both MDS-derived and wild-type BFU-E, as well as a lack of MDS-derived maturing erythroblasts, indicating that wild-type erythroblasts compensate for an intrinsic defect in clonal erythropoiesis. The progressive development of anemia in NHD13 chimeric mice was associated with failure of wild-type erythroid precursor expansion in the marrow, implicating extrinsic, environmental factors as the cause of anemia. We replicated these results in a complementary, EVI1 overexpression chimeric mouse model of MDS/marrow failure. Anemia in NHD13 chimeric mice was also associated with expansion in the marrow of MDS clone-derived monocytes, which expressed higher levels of several inflammatory cytokines, compared with monocytes from age-matched control mice transplanted with wild-type cells. We also found increased erythroblast-monocyte interactions by imaging flow cytometry, consistent with alterations in the erythroblastic island microenvironment. Taken together, these data indicate that the development of anemia in two independent murine models of MDS is due not only to an intrinsic block of MDS clonal erythropoiesis, but also to an extrinsically-mediated failure of residual, predominantly wild-type, erythropoiesis.

The Spliceosomal Component Sf3b1 Is Essential for Hematopoietic Differentiation

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. A multitude of recurrent somatic mutations in spliceosomal components were recently identified in MDS that likely contribute to the pathogenesis of the disease. The lack of in vivo models to study cell-type specific effects of spliceosomal mutations limits our understanding of why such mutations lead to hematopoietic abnormalities. Using a zebrafish with a loss-of-function mutation in sf3b1 (sf3b1hi3394), an essential member of the spliceosome, we demonstrate hematopoietic cell differentiation and hematopoietic stem and progenitor cell (HSPC) specification are processes sensitive to spliceosomal malfunction. Primitive erythropoiesis initiates normally in sf3b1 mutants as evidenced by expression of scl in the posterior lateral plate mesoderm at 14 hours post fertilization (hpf) as well as gata1 and beta-globin at 24 hpf. Flow cytometry quantification of gata1:gfp positive erythrocytes showed sf3b1 mutants have 25% more cells at 24 hpf, but greater than 3-fold fewer cells at 36 and 48 hpf, time points when wild type erythroblasts are expanding and differentiating. At 48 hpf, we also observed decreased levels of o-dianisidine positive erythrocytes, low numbers of morphologically mature erythroblasts, and higher levels of immature erythroblasts in sf3b1 mutants. Similarly, we observed normal initiation of primitive myelopoiesis marked by stem cell leukemia (scl) expression in the anterior lateral plate mesoderm at 14 hpf, but diminished expression of more differentiated markers, l-plastin and myeloperoxidase at 24 and 28 hpf in sf3b1 mutants. Quantification of lysozyme C:dsred positivemyeloid cells using flow cytometry also showed 24-fold fewer mature myeloid cells in sf3b1 mutants at 36 hpf. Our data on primitive erythropoiesis and myelopoiesis indicate sf3b1 is required for hematopoietic cell differentiation. Additionally, sf3b1 mutants have diminished expression of the definitive HSPC marker runx1 within the aorta at 28 hpf. In contrast, we observed normal expression of the pan-endothelial marker kinase insert domain receptor-like (kdrl) and aorta-specific markers notch1b and notch3 at 24 hpf. Flow cytometry quantification of kdrl:gfp endothelial cells at 24 hpf shows no difference in the frequency of endothelial cells in sf3b1 mutants. Moreover, we observed fewer cmyb:gfp; kdrl:dsred double positive HSPCs along the dorsal aorta. The data suggest that decreased HSPC formation in sf3b1 mutants is due to a failure in hemogenic induction. From these studies, we show sf3b1 is required at specific stages of hematopoietic cell development. These results provide novel insight into the role of splicing in blood cell development, and can afford a deeper understanding of the mechanism of splicing regulation on the origins of MDS. DisclosuresNo relevant conflicts of interest to declare.

Loss of Foxo3 Reduces Erythroblast Apoptosis and Enhances RBC Production in Beta-Thalassemic Mice

β-thalassemia arises as a result of mutations in the β-globin gene. As a consequence erythropoiesis, the process that insures the daily generation of billions of red blood cells (RBCs), becomes disrupted. Ineffective erythropoiesis is a major contributor to the β-thalassemic anemia and is partially due to aberrant apoptosis during late stages of erythroid maturation. Despite the importance of apoptosis, the underlying molecular mechanisms regulating this process in β-thalassemia erythroblasts are not fully elucidated. One potential mechanism involves the transcription factor Foxo3, which under specific contexts can act as a positive regulator of apoptosis, but is also an essential transcriptional regulator of terminal erythroblast maturation. Foxo3 has a range of outputs that it can execute from sustaining cellular integrity by mitigating oxidative stress to inducing apoptosis under conditions of overwhelming stress. Given these functions, we sought to determine if Foxo3 played a role in maintaining RBC maturation in β-thalassemic mice. To address this, we used Hbbth3/+ (th3/+) mice that display a phenotype similar to β-thalassemia intermedia, and produced double mutant Foxo3-/-/Th3/+ mice. The th3/+ mice display a mild erythroblast apoptotic phenotype. We hypothesized that loss of Foxo3 may exacerbate the β-thalassemic phenotype. On the contrary, we found that loss of Foxo3 in a β-thalassemic background improved RBC numbers and hemoglobin concentration (by 1g/dl, n=10 mice) in double mutant mice compared to th3/+ mice. Furthermore, double mutant mice had a statistically significant lower frequency of apoptosis (2 fold less) during bone marrow erythroblast maturation as measured by flow cytometry analysis of annexin V-binding and 7AAD staining in distinct erythroblast stages resolved by TER119, CD44 and cell size (n=3 mice per genotype). We predicted that high levels of oxidative stress may prematurely activate FOXO3 during erythroblast maturation in β-thalassemic mice. In turn, activated FOXO3 may potentially promote apoptosis in these cells. To evaluate this, we examined FOXO3 levels by qRT-PCR and immunofluorescence in FACS sorted populations of erythroblasts (TER119+,CD44,FSC) or erythroid progenitors (TER119-,c-KIT+,CD71HI) acquired from bone marrow of at least 3 mice per genotype. Our data show increased mRNA levels of Foxo3 in early erythroblasts, corresponding to increased FOXO3 protein expression in erythroid progenitors from β-thalassemic mice relative to wild-type mice. We also examined the activation status of p53, as it is also a major regulator of apoptosis that can be triggered by oxidative stress. Nuclear p53 levels were greater in β-thalassemic as compared to wild-type erythroid progenitors based on immunofluorescence analysis of sorted cells from bone marrow of 3 mice per genotype. These results suggest a higher level of active p53 in β-thalassemic erythroid progenitors. Our results provide evidence that FOXO3, a factor normally critical for erythroblast maturation, may cooperate with aberrantly active p53 to induce apoptosis in β-thalassemic erythroblasts. In support of this, downstream p53 targets including Gadd45a and p21 that are also Foxo3 targets were significantly upregulated in β-thalassemic erythroblasts relative to wild-type erythroblasts as determined by qRT-PCR of cDNA produced from 3 mice per genotype. To more closely examine the mechanism of decreased apoptosis in double mutant Foxo3-/-/Th3/+ erythroblasts, we compared the expression of multiple genes involved in apoptosis by qRT-PCR of sorted erythroblast populations from at least 3 mice per genotype. We found multiple pro-apoptotic genes including, Cycs, Tnfsf10, Puma, and Bim expressed at significantly lower levels at various erythroblast stages in double mutant compared to β-thalassemic erythroblasts. Together, our data suggests Foxo3 becomes inappropriately and prematurely activated in erythroid progenitors and early erythroblasts in the context of β-thalassemia and cooperates with p53 to promote apoptosis. These findings raise the possibility that cooperation of Foxo3 and p53 in β-thalassemic erythroblasts might contribute to the ineffective erythropoiesis of β-thalassemic mice. They also suggest the possibility that as a homeostatic maintaining factor, Foxo3 behaves differently in the context of disease. Disclosures No relevant conflicts of interest to declare.

Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation

AbstractRhoA GTPase has been shown in vitro in cell lines and in vivo in nonmammalian organisms to regulate cell division, particularly during cytokinesis and abscission, when 2 daughter cells partition through coordinated actomyosin and microtubule machineries. To investigate the role of this GTPase in the rapidly proliferating mammalian erythroid lineage, we developed a mouse model with erythroid-specific deletion of RhoA. This model was proved embryonic lethal as a result of severe anemia by embryonic day 16.5 (E16.5). The primitive red blood cells were enlarged, poikilocytic, and frequently multinucleated, but were able to sustain life despite experiencing cytokinesis failure. In contrast, definitive erythropoiesis failed and the mice died by E16.5, with profound reduction of maturing erythroblast populations within the fetal liver. RhoA was required to activate myosin-regulatory light chain and localized at the site of the midbody formation in dividing wild-type erythroblasts. Cytokinesis failure caused by RhoA deficiency resulted in p53 activation and p21-transcriptional upregulation with associated cell-cycle arrest, increased DNA damage, and cell death. Our findings demonstrate the role of RhoA as a critical regulator for efficient erythroblast proliferation and the p53 pathway as a powerful quality control mechanism in erythropoiesis.

Argonaute HITS-CLIP Reveals Global miRNA-mRNA Networks in Erythropoiesis

MicroRNAs (miRNAs) inhibit gene expression by recruiting the RNA-induced silencing complex (RISC) to specific sets of target mRNAs. However, it has been challenging to define precisely the miRNA-target mRNA interactions that occur within cells. One approach to rigorously characterizing these networks in cells of interest is to sequence mRNA fragments bound to the RISC component Argonaute using a method termed High-Throughput Sequencing of RNA isolated by Crosslinking Immunoprecipitation (HITS-CLIP). We used this approach to define miRNA-target mRNA interactions during erythropoiesis, a developmental process known to be regulated by miRNAs and dramatically impaired by loss of Argonaute function.We performed Argonaute HITS-CLIP on primary mouse fetal liver erythroblasts in 3 biological replicate experiments and used Bowtie tools to map the sequenced RNA fragments to the mouse genome. These sequences represent mature miRNAs and their bound RNA segments in erythroid cells. Remarkably, miR-451 accounted for 70% of the Ago-bound erythroid miRNA burden, with miR-142, miR-144, miR-21, miR-374 and miR-30 accounting for an additional 15%. The frequency of Ago-binding correlated well with total cellular miRNA abundance. To analyze miRNA-target interactions, we used Piranha tools to identify peaks of sequenced reads, focusing on those that map to protein coding mRNAs. We identified 3,670 peaks across the entire genome, most of which mapped to mRNA 3’ untranslated regions (UTRs) (45%) or protein coding regions (38%). The remaining peaks mapped to intergenic regions, introns, long noncoding RNA genes and pseudogenes.To identify miRNA-mRNA associations, we searched for base pair complementarity between HITS-CLIP peaks mapping to mRNAs and seed sequence matches to the top 20 miRNAs in erythroid cells, which account for 92% of total miRNAs. We matched 36% of all peaks to canonical seed sequences and 34% to non-canonical seed sequences of specific miRNAs. We compared miR451-target mRNA matches predicted by HITS-CLIP to mRNA expression datasets generated from wild-type (WT) and miR-144/451 knockout (KO) mouse fetal liver erythroblasts. mRNAs with canonical miR-451 peaks in 3’ UTRs were significantly upregulated in KO erythroblasts as compared to WT erythroblasts (p<0.0001), indicating that our HITS-CLIP study identifies true erythroid targets of miR-451. These include previously identified targets of miR-451 such as Cab39, Ywhaz and Vapa, further validating our study. We also identified previously unknown targets such as Copa and Reep5, which, along with Vapa, regulate vesicle trafficking in other cell systems, and Matr3, Patl1 and Ythdf2, which affect global mRNA stability. Study of these genes may provide further insight into the functions of miR-451 in erythropoiesis. In contrast, mRNAs with predicted miR-451 peaks within coding exons showed no change in expression. This indicates that not all Ago HITS-CLIP peaks reflect regulation of mRNA stability, and that the presence of 3’ UTR Ago peaks with canonical miRNA seed sequences predicts better this mode of post transcriptional control of gene expression. Analysis and comparison of mRNA translational regulation by 3’UTR and coding region-bound miRNAs is in progress.Overall, our results provide a comprehensive map of Ago-bound miRNAs and their biologically relevant target mRNAs in erythroid cells. This map will serve as a basis to better understand miRNA regulation of erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Targeted Gene Disruption of Kinesin-3 Family Motors Unveils a Negative Regulatory Mechanism for Mouse Erythroblast Enucleation

Abstract 371 [Display omitted] Terminal differentiation of mammalian erythroid precursors involves enucleation, a process required for the production of reticulocytes. The signaling pathways and the molecular components mediating the final enucleation step are not well understood, although the resemblance of enucleation to asymmetric cell division has been suggested. The enucleation of erythroblasts can be replicated in vitro; however, the enucleation efficiency is not optimal under in vitro culture conditions. A functional role of both microfilaments and microtubules has been suggested in the regulation of erythroblast enucleation. We hypothesized that molecular motors known to regulate asymmetric cell division might also play a functional role in erythroblast enucleation. GAKIN (also called KIF13B) is a kinesin-3 motor implicated in the regulation of cell polarity pathways. Its Drosophila homologue Khc-73 regulates polarity formation during the asymmetric cell division of neuroblasts. We generated a mutant mouse line of GAKIN and examined the efficiency of erythroblast enucleation using the fetal liver in vitro erythroid precursor culture system originally developed by the Lodish group. The GAKIN mutant mouse line was produced from the embryonic stem (ES) cells containing a genomic insertion of the beta-galactosidase-neomycin gene. The beta-geo insertion was mapped within intron 38 of the GAKIN gene, thus removing the CAP-Gly domain located at the C-terminus of GAKIN. This allowed us to visualize specific tissue expression of GAKIN, in addition to the subcellular localization of a GAKIN-lacZ fusion protein by lacZ activity staining. The lacZ activity revealed that the GAKIN-lacZ fusion protein is expressed in bone marrow macrophages and erythroblasts, while also highly concentrated at contact sites between macrophages and erythroblasts. GAKIN mutant mice are viable and fertile, and complete blood analysis did not reveal any discernible phenotype. To test for the enucleation efficiency, erythroid precursors were isolated from fetal liver stage at day 14.5 embryos and enucleation was quantified by flow cytometry after 2 days of in vitro culture. The GAKIN-lacZ fusion protein appeared as a single dot representing the microtubule organizing center (MTOC) in the erythroid precursors isolated from fetal liver (Fig. 1). However, GAKIN mutant erythroblasts enucleated at the same efficiency as wild type erythroblasts under steady state conditions in vitro. Since the mammalian genome contains a close homologue of GAKIN/KIF13B, termed KIF13A, a possibility exists that KIF13A functionally compensates for GAKIN mutation in critical pathways. To address this issue, we generated KIF13A null mice using a similar gene disruption strategy. Again, the KIF13A mutant erythroblasts did not show any measurable change in enucleation efficiency under similar conditions. However, fetal liver erythroblasts isolated from GAKIN and KIF13A double mutant mice exhibited a significant enhancement of enucleation efficiency. This finding suggests that together, GAKIN and KIF13A negatively regulate the erythroblast enucleation by modulating the microtubule-based cytoskeleton. We propose that efficient erythroid enucleation in vivo involves signaling mechanisms inhibiting both GAKIN and KIF13A motors. Since erythrocytes from adult double mutant mice appear to be hematologically normal, our findings raise the possibility of enhancing the in vitro production of functional erythrocytes by inhibiting GAKIN and KIF13A activity in CD34 positive stem cells. Disclosures:No relevant conflicts of interest to declare.

Epo/Epor Signaling Critically Regulates Maturation of the Primitive Erythroid Lineage In the Mammalian Embryo

AbstractAbstract 813Erythropoiesis in both the embryo and the adult is characterized by the progressive maturation of lineage-specific progenitors through morphologically identifiable precursors that enucleate to form mature erythrocytes. In the adult, erythropoietin (Epo) binds its homodimeric receptor (EpoR) and activates the Jak2-Stat5 pathway to inhibit apoptosis and mediate the proliferation of definitive erythroid progenitors. Similar responses to Epo have also been reported in neurons, cardiomyocytes, and several cancer cell types. However, little is known regarding the function of Epo/EpoR in the first erythroid cells of the developing embryo, the yolk sac-derived, primitive erythroblasts that emerge from a transient wave of progenitors and mature as a synchronous cohort in the fetal vasculature. We have previously determined that EpoR transcripts are present in yolk-sac blood islands beginning at E7.5 (McGann et al., Exp Hem, 1997). Here, we examine the function of Epo/EpoR in embryonic erythropoiesis. Using a colony-forming assay, we found normal levels of primitive erythroid progenitor activity in E8.5 Epor-null embryos. However, these colonies were composed of markedly fewer cells, suggesting a defect in the erythroid precursor maturation. Consistent with these in vitro results, we found that E10.5 Epor-null embryos contain 60% fewer primitive erythroblasts compared to wild-type littermates. Cell cycle analysis revealed a block at the transition from G1- to S-phase with a greater than 2-fold increase in the proportion of erythroblasts in G1-phase in Epor-null versus wild-type embryos. Furthermore, utilizing multi-spectral imaging flow cytometry, we found that E10.5 and E11.5 Epor-null primitive erythroblasts exhibit decreased cell size, nuclear size, and RNA content. Transcriptional analysis revealed a higher εy:βH1 expression ratio in Epor-null erythroblasts consistent with a more rapid maturational globin switch. Most strikingly, we detected a 10-fold reduction in CD71 (transferrin receptor) cell surface expression, which led us to investigate its transcript levels along with other known Stat5-target genes. CD71, Myc, and Bcl-xl transcript levels were all reduced significantly in Epor-null compared to wild-type erythroblasts. Furthermore, we found robust phosphorylation of Stat5 in primitive erythroid precursors that increased upon exposure to Epo. Taken together, our findings indicate that Epo/EpoR signaling is not required for the specification or emergence of primitive erythroid progenitors. However, the subsequent proliferative maturation of primitive erythroid precursors is highly dependent on Epo/EpoR signaling and modulation of Stat5-target genes. The marked reduction of primitive erythroid precursors in Epor-null embryos is associated with cell cycle defects. Furthermore, Epor-null primitive erythroblasts exhibit a phenotype of premature maturation, characterized by decreased cell size, nuclear size, RNA content, and altered globin switching. Finally, the drastic reduction of CD71 expression supports the concept that Epo regulates iron metabolism through Stat5-mediated signaling. We conclude that erythropoietin is a critical regulator of primitive erythropoiesis in the mammalian embryo. However, unlike definitive erythropoiesis, Epo/EpoR primarily regulates the primitive lineage at the precursor rather than the progenitor stage of maturation. Disclosures:No relevant conflicts of interest to declare.

A Screen In Primary Erythroblasts Reveals a MicroRNA-Centered Homeostatic Mechanism for Regulating Cyclin E Activity.

AbstractAbstract 1543A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin ET74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin ET74A T393A knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3' untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCFFbw7 ubiquitin ligase can be controlled by the microRNA pathway. Disclosures:No relevant conflicts of interest to declare.

Functional Analysis of Transcription Factor c-Maf in Embryonic Hematopoiesis.

Abstract 3646Poster Board III-582c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the AP1 super family of basic leucine zipper proteins. c-Maf has been shown to be important for the regulation of IL-4 and IL-21 gene expression in T-helper-2 cells and the formation of the lens structure. A recent report has demonstrated that the augmentation of c-Maf gene expression was observed in myeloma cell lines and bone marrow samples of multiple myeloma patients. It suggests that c-Maf plays important role in multiple myeloma pathogenesis. However, the physiological roles of c-Maf in normal hematopoiesis are largely unknown and have never been analyzed in vivo setting. In this study, we investigated that c-Maf is indispensable for hematopoesis via analyzing c-Maf deficient (c-Maf−/−) mice. In the C57BL/6J background, c-Maf−/− mice were embryonic lethal and it died between embryonic day (E) 15 and E18. c-Maf−/− embryo looked pale and had evidence of anemia. In c-Maf−/− peripheral blood at E14.5, the number of enucleated erythrocytes was decreased compared that in wild-type (WT) littermates. Flow cytometric analysis of fetal liver cells indicated that mature red blood cells were reduced in c-Maf−/− embryos. In addition, Annexin-V positive apoptotic cells were increased in c-Maf−/− fetal liver. Interestingly, there was no significant difference between WT and c-Maf−/− fetal liver cells in erythroid colony counts on the colony-forming unit assay. These results indicated that c-Maf−/− erythroid cells can develop to mature cells, and the impaired definitive erythropoiesis in the c-Maf−/− mice is due to a non-cell-autonomous effect. Immunohistochemical staining of WT fetal liver using anti-c-Maf antibody showed that c-Maf was highly expressed in macrophage but not in erythroid cells. For this reason, we hypothesized that c-Maf−/− macrophage was responsible for anemia of c-Maf−/− embryo. The erythropoietic microenvironment in fetal liver mainly consists of clusters of erythroblastic islands that made up of central macrophage and surrounding erythroblasts. The compromise of erythroblasitc islands through defect of macrophage causes anemia and embryonic lethality. To verify whether c-Maf−/− macrophage cause fail to form erythroblasitic island, we isolated the erythroblasitc island from WT and c-Maf−/− fetal liver. As a result, the number of erythroblast surrounding the macrophage in erythroblastic island was significantly reduced in c-Maf−/− mice. Moreover, in vitro reconstitution experiments showed that c-Maf−/− erythroblasts were able to form erythroblastic island with WT macrophages, while c-Maf−/− macrophages and WT erythroblasts could not form erythroblastic island. These results strongly suggest that the cause of anemia in c-Maf−/− mice is due to abnormal function of macrophages in forming erythroblastic island. Finally, to clarify the molecular mechanism in formation of erythroblastic island, we found the target gene of c-Maf in fetal liver macrophages. The DNA microarray and real time RT-PCR analyses showed that the decreased expression of vascular adhesion molecule 1 (VCAM-1), known as an adhesion molecule expressed on macrophage of erythroblastic island, was observed in c-Maf−/− fetal liver macrophage. We conclude that c-Maf is indispensable for definitive hematopoiesis and erythroblastic island formation through crucial roll in fetal liver macrophages. Disclosures:No relevant conflicts of interest to declare.

Deregulated E2f-2 Underlies Cell Cycle and Maturation Defects in Retinoblastoma Null Erythroblasts

By assessing the contribution of deregulated E2F activity to erythroid defects in Rb null mice, we have identified E2f-2 as being upregulated in end-stage red cells, where we show it is the major pRb-associated E2f and the predominant E2f detected at key target gene promoters. Consistent with its expression pattern, E2f-2 loss restored terminal erythroid maturation to Rb null red cells, including the ability to undergo enucleation. Deletion of E2f-2 also extended the life span of Rb null mice despite persistent defects in placental development, indicating that deregulated E2f-2 activity in differentiating erythroblasts contributes to the premature lethality of Rb null mice. We show that the aberrant entry of Rb null erythroblasts into S phase at times in differentiation when wild-type erythroblasts are exiting the cell cycle is inhibited by E2f-2 deletion. E2f-2 loss induced cell cycle arrest in both wild-type and Rb null erythroblasts and was associated with increased DNA double-strand breaks. These results implicate deregulated E2f-2 in the cell cycle defects observed in Rb null erythroblasts and reveal a novel role for E2f-2 during terminal red blood cell differentiation. The identification of a tissue-restricted role for E2f-2 in erythropoiesis highlights the nonredundant nature of E2f transcription factor activities in cell growth and differentiation.

Bcl-xL Does Not Rescue Erythroid Colony (CFU-e) Formation in EpoR−/− Progenitors, Suggesting a Cell-Cycle Role for EpoR.

The essential role of glycoprotein hormone erythropoietin (Epo) and its receptor, EpoR, in erythroid development is well established: both the EpoR−/− and Epo−/− mouse embryos die on embryonic day 13 (E13) due to failure of definitive erythropoiesis in fetal liver (Wu et al. 1995). It has been suggested that Epo's principal role during erythropoiesis is to protect erythroid progenitors from apoptosis (Koury and Bondurant, Science 1990). Bcl-xL, an anti-apoptotic member of the bcl-2 family, is induced by EpoR signaling in erythroid cells via the Jak2/Stat5 pathway (Silva et al., Blood 1996; Socolovsky et al., Cell, 1999). Bcl-xL is essential for erythroid maturation: bcl-xL−/− embryos die in utero at the same stage as as EpoR−/− mice, lacking definitive erythropoiesis (Motoyama et al., Science 1995; J Exp Med, 1999). Recenlty, it has been shown that over-expression of bcl-xL in primary wild-type erythroblasts confers Epo independence on these cells in vitro and allows them to complete their differentiaion into red blood cells (Dolznig et al., Curr Biol, 2002). Here we reasoned that if the principal function of EpoR signaling is suppression of apoptosis via bcl-xL, it should be possible to rescue all aspects of erythroid differentiation in EpoR−/− fetal liver progenitors by retrovirally-transducing these cells with bcl-xL.We infected EpoR−/− fetal liver progenitors with bicistronic retroviral vectors expressing either bcl-xL or EpoR, each linked via an IRES sequence to a GFP reporter. Control EpoR−/− cells were infected with ‘empty' bicistronic vector. Infection rates were in excess of 30% for all constructs, and transduced cells were identified for further analysis using GFP fluorescence. We examined terminal differentiation of the transduced EpoR−/− cells over the ensuing 48 hours, using several distinct assays, including their expression of the cell-surface differentiation markers CD71 and Ter119 by FACS, their ability to give rise to CFU-e colonies in semi-solid medium, their cell-cycle status using DNA content analysis and BrdU incorporation, and their maturation and hemoglobinization by diaminobenzidine staining and light microscopy.We found that EpoR−/− progenitors transduced with bcl-xL were protected from apoptosis, and underwent morphological changes characteristic of erythroid maturation, including decreasing cell size, nuclear condensation and expulsion, and accumulation of hemoglobin. These cells also upregulated the erythroid-specific cell surface marker Ter119. However, unlike EpoR−/− cells transduced with EpoR, bcl-xL -transduced cells did not express high levels of CD71, and failed to give rise to CFU-e colonies in semi-solid medium. Instead, they gave rise to small colonies of 6 cells or less. Cell cycle analysis showed that, throughout the 48 hours of erythroid terminal differentiation, the population of bcl-xL-transduced EpoR−/− cells had a lower fraction of cells in S-phase than control, EpoR-transduced EpoR−/− cells. The cell-cycle status of control, terminally-differentiating wild-type erythroid fetal liver progenitors was not altered by transduction with bcl-xL, excluding the possibility that it directly inhibits S-phase.Taken together our results indicate that bcl-xL does not rescue all aspects of erythroid differentiation in EpoR−/− erythroid progenitors. Specifically, the proliferative program during erythroid terminal differentiation is directly dependent on EpoR signaling, and is not simply a default pathway secondary to EpoR's anti-apoptotic effect.

Differential gene expression profiling between wild-type and ALAS2-null erythroblasts: Identification of novel heme-regulated genes

To identify erythroid-specific heme-regulated genes, we performed differential expression analysis between wild-type and heme-deficient erythroblasts, which had been prepared from wild-type and erythroid-specific δ-aminolevulinate synthase-null mouse ES cells, respectively. Among 8737 clones on cDNA array, 40 cDNA clones, including 34 unknown ESTs, were first selected by their high expression profiles in wild-type erythroblasts, and evaluated further for their erythroid-lineage specificity, expression in hematopoietic tissues in vivo, and heme-dependent expression, which yielded 11, 4, and 4 genes, respectively. Because of the selection strategy employed, the final 4 were considered as the newly identified erythroid-specific heme-regulated genes. These 4 genes were uncoupling protein 2, nucleolar spindle-associated protein, cellular nucleic acid-binding protein, and a novel acetyltransferase-like protein. These findings thus suggest that heme may regulate a wide variety of hitherto unrecognized genes, and further analysis of these genes may clarify their role in erythroid cell differentiation.

Transferrin Receptor 1 Up-Regulation Plays a Role in Iron Accumulation in a Murine Model of Sideroblastic Anemia.

Manganese superoxide dismutase (SOD2) detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973). While SOD2-deficiency is lethal, SOD2-deficient (SOD2−/−) hematopoietic stem cells can rescue lethally irradiated wild-type mice. SOD2−/− hematopoietic chimeras show a persistent hemolytic anemia similar to human sideroblastic anemia (Friedman et al. J. Exp. Med. 2001). SOD2−/− erythroid progenitor cells have increased mitochondrial mass, and reticulocytes show mitochondrial iron deposition. Mature RBC show abundant siderotic granules, evidence of a defect in iron incorporation into heme, and shortened lifespan. SOD2−/− progenitors and mature RBC show both enhanced reactive oxygen species production and protein oxidative damage (Friedman et al. Blood 2004; Martin et al. Submitted).To define early events in the pathogenesis of the SOD2-deficiency anemia and, in particular to identify genes involved in the response of erythroid progenitors to oxidative stress, we compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2−/−versus wild-type hematopoietic stem cells recipients. Using cDNA microarrays and class comparison analysis, we identified 600 transcripts as significantly discriminant between genotypes. Analysis showed that eleven transcripts encoding different subunits of the mitochondrial oxidative phosphorylation, ATP synthase, and TCA cycle were down-regulated in SOD2−/− erythroblasts. Previous work showed similar results at the protein level in SOD2−/− RBC (Friedman et al. Blood 2004) and at the activity level in specific tissues of SOD2−/− neonates prior to death (Melov et al. PNAS 1999). One interpretation is that SOD2−/− erythroblasts show metabolic decline. Of interest, a single transcript involved in iron homeostasis, Trfr, was found to be expressed at twice the levels found in wild-type erythroblasts (p<0.0007, parametric p value). Trfr encodes transferrin receptor 1; two-fold up-regulation of transferrin receptor 1 was also shown at the protein level by flow cytometry analysis of SOD2−/− erythroblasts (p<0.0001, unpaired two-tailed t-test). Transferrin receptor 1 is the cellular gatekeeper for iron uptake whose genetic inactivation induces abnormal erythropoiesis and iron homeostasis (Levy et al. Nat. Genet. 1999). The stability of the Trfr transcript is highly regulated by iron regulatory proteins (IRPs), that are known to be controlled by numerous effectors including reactive oxygen species (Hentze et al. Cell 2004, for review). We focus our current work on investigating, in vitro and in vivo, the role that up-regulation of transferrin receptor 1, likely through oxidative stress-mediated IRPs activity regulation, plays in iron overload in our SOD2-deficiency model.Taken together, our findings raise the possibility that increased iron delivery may be sufficient to cause sideroblastic anemia or may contribute to a self-reinforcing cycle where oxidative stress favors increased iron, and increased iron results in enhanced oxidative damage.

Aberrant iron accumulation and oxidized status of erythroid-specific δ-aminolevulinate synthase (ALAS2)–deficient definitive erythroblasts

AbstractAlas2encodes the erythroid-specific δ-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with theAlas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking theAlas2gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype ofAlas2-null definitive erythroblasts was examined.Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes inAlas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate thatAlas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron,Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.