ALBERT

Description: Abstract 683 Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that 90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis. Disclosures: No relevant conflicts of interest to declare.

Description: MicroRNAs are ~22nt-long small noncoding RNAs that negatively regulate expression of target proteins through mRNA degradation or translational repression. They are functionally important in diverse biological processes including development, oncogenesis, and hematopoiesis. To study a potential role of microRNA for regulating cellular changes during erythroid ontogeny, we examined microRNA abundance patterns in circulating erythroid cells during the fetal and adult stages of human development. Expression profiling of microRNA was performed using total RNA from 4 adult peripheral blood samples compared to 4 cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNA was hybridized to custom spotted array containing 474 human microRNAs species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines was used as a hybridization reference for all samples to generate microRNA abundance profile for each sample. Among the differentially expressed probe-pairs, 40 microRNAs were significantly up-regulated in adult erythroid cells and only 4 microRNAs were down-regulated (p 〈 0.01 and fold change 〉 2). Two microRNAs, miR-144 and miR-451, previously reported as being GATA-1 regulated in erythroid cells, were detected above the dynamic range of the array technology in all eight samples. Quantitative PCR was performed to further quantitate differences in the microRNA abundance profiles of the 10 most differentially expressed microRNAs as well as miR-144 and miR-451. Per published recommendations, miR-103 was used for signal normalization. Among the studied microRNAs, 8 of 12 demonstrated a more than 4 fold increase in abundance in adult erythroid cells compared to cord blood by qPCR. In particular, let-7d and let-7e demonstrated more than 10-fold increased expression in adult cells. Of note, let-7 microRNAs regulate metamorphic processes in model organisms and inhibit ras-associated growth of cancer cells. According to miRBase predictions, over 1200 genes may be targeted by the 8 microRNAs (p 〈 0.001). Examination of cord and adult reticulocyte mRNA expression profiles (Goh et al. Phy. Gen. 2007) revealed 24 developmentally downregulated genes as predicted targets. Among globin genes, only gamma globin is a predicted target. These findings demonstrate dramatic up-regulation of specific erythroid microRNAs during the fetal-to-adult transition of ontogeny, and support the notion that microRNAs function as developmental regulators of erythropoiesis and globin gene switching.

Description: Alterations of globin-gene cluster expression mediated through cytokine signal transduction have been previously established. In adult erythroblasts, ex vivo combinatorial signaling from cytokines such as erythropoietin (EPO), stem cell factor (SCF), and transforming growth factor-beta (TGF-B) causes a robust reactivation of fetal hemoglobin. To determine if cytokine-activated expression of fetal hemoglobin significantly alters the transcript and protein abundance of nuclear transcription factors, profiling studies of transcription factor expression were performed. Primary CD34+ cells were cultured as donor-matched pairs using cytokine combinations that produced low vs. high levels of gamma-globin mRNA and fetal hemoglobin (Bhanu et al. Blood, 2005). To identify candidate genes, total RNA from 15 separate healthy volunteer donors was collected and combined into 5 pools. The pooled RNA was labeled and hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips. Informatics strategies were aimed toward identifying differential expression of those transcription factors with binding motif on any of the 10 human globin gene promoter regions (553 transcription factors; 115 binding motif families), versus all transcription factors demonstrating robust expression with changes of at least 3-fold. Combining both strategies, 11 candidate transcription factors were identified (BCL11A, CBFB, EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, SOX-6, TCF3, and THRB). RNA-based descriptions of gene activity do not necessarily reflect changes in protein levels; therefore, Western analyses were performed. GATA-1 was utilized as a control, since no significant change in GATA-1 mRNA was detected by expression array profiling. Nuclear protein extracts were obtained from 3 additional donors’ CD34+ cells cultured under conditions of low vs. high gamma-globin mRNA synthesis (1.6E+06 ± 3.8E+05 and 1.5E+07 ± 6.3E+06 copies/ng total RNA, respectively). Western blot analysis revealed reproducible and robust differences in protein expression levels for 7 of the 11 candidate transcription factors (EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, and SOX-6). Two other candidates were expressed below the protein detection limit. GATA-1 and the remaining 2 candidates demonstrated no change in the nuclear protein levels. Among the group with confirmed changes in gene expression, ELK-1 and EGR-1 are downstream targets of the MAP kinase signal transduction cascade. MAFF, ID2, and SOX-6 are known regulators of globin gene expression. The differential expression of HHEX and MNDA warrants further investigation. These data demonstrate that cytokine signal transduction causes changes in the intranuclear levels of at least 7 transcription factors concurrently with activation of gamma-globin gene expression.

Description: Abstract 4255 Based upon the importance of iron for erythropoiesis, it was hypothesized that specific molecular and cellular mechanisms may be manifested during early erythropoiesis in preparation for heme and hemoglobin production. Here iron regulation was studied in immature erythroid progenitor cells prior to the increased production of heme or hemoglobin. CD34+ cells were cultured in SCF, IL-3 and FLT-3 ligand for 7 days, and the erythroid progenitor cells were identified and sorted from three donors according to the differential expression of CD36, CD41, and CD71. Among the sorted populations, the CD71(+)CD36(+)CD41(-)GPA(-) cells grew almost exclusively (〉95%) into erythroid colonies in methylcellulose culture. Cytospin examination of those erythroid progenitor cells demonstrated large blasts with deep blue cytoplasm and blebbed membranes consistent with pre-proerythroblasts. Furthermore, heme accumulation and hemoglobin were absent. Western analyses revealed that ferritin, transferrin receptor 1 (TfR1), and transferrin receptor 2 (TfR2) expression were regulated by dosed titrations (0, 10, 20, 40, 60, and 100 %) of iron-saturated transferrin in the culture medium. The intracellular iron-storage protein, ferritin, showed increased expression with higher holo-transferrin saturation. Opposite patterns of iron-dependent regulation for the transferrin receptors was identified. TfR1 protein decreased and TfR2 increased with increased transferrin saturation. While TfR1 was detected at high levels on the plasma membranes, immunofluorescence studies showed TfR2 co-localization with lysosomes (correlation with LAMP1, a lysosomal marker, R = 0.53 ± 0.11, n=22). Decreased holo-transferrin in the culture medium resulted in no significant reduction in the proliferation of these cells until hemoglobin production was induced by erythropoietin. Confocal microscopy demonstrated a distinct peri-centrosomal lysosomal compartment surrounded by mitochondria. Coincidentally, upon sulfide-silver staining, it was observed that iron accumulated within the lysosomal compartment in a transferrin-dependent fashion. No similar iron-containing lysosomal-mitochondrial structure was identified in non-erythroid cells in the same cultures. These results suggest that functional, lineage-specific differentiation occurs at the progenitor stage of hematopoiesis, and that iron import and regulation is highly developed during erythropoiesis prior to hemoglobin production. Disclosures: No relevant conflicts of interest to declare.

Description: Iron deficiency affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. However, iron depleted erythropoiesis is incompletely understood. In this study, an ex vivo model of iron depleted erythropoiesis was developed using dosed titrations of the iron chelator deferoxamine (DFO) in CD34+ cell cultures. All experiments were performed in triplicate with cells from three separate donors. Hemoglobinization and expression patterns of erythroid markers (GPA/CD71) demonstrated minimal changes in DFO supplemented medium. DFO caused dose-related suppression of cell counts after 14 days (20uM DFO: 42% reduction, 30uM DFO: 86% reduction, 40uM DFO: 96% reduction of cell counts compared to 0uM DFO controls). Cultures supplemented with 30uM DFO were used for subsequent studies, and the DFO-mediated effects were completely reversed by addition of 30uM ferric chloride. To investigate whether apoptosis caused the reduction in cell counts, surface Annexin V was measured by flow cytometry. No significant increases in levels of apoptosis among the proliferating erythroblasts were detected (Annexin V positive cells; 0uM DFO 6.2 ± 0.95%, 30uM DFO 9.0 ± 1.55%, p = 0.058). Analyses of p53 protein quantitation and p53 DNA-binding activity further suggested that the suppression of cell counts by iron chelation was not due to p53 related apoptosis. In the absence of apoptosis or maturation arrest, we hypothesized that the growth suppressing effects of DFO were not due to ineffective erythropoiesis. To test this hypothesis, a recently-discovered marker of ineffective erythropoiesis named GDF15 (Tanno et al. Nat. Med. 2007) was measured in the culture supernatants, and no increase was detected (GDF15 concentration per 1×104 cells: 0uM DFO; 79.7 ± 4.4 pg, 30uM; 43.9 ± 4.3 pg). In the clinic, serum GDF15 levels from 17 blood donors with significantly reduced ferritin and iron saturation (279 ± 98 pg/ml) were not increased when compared to serum GDF15 levels in 17 healthy volunteers with normal iron parameters (410 ± 119 pg/ml). To determine if cell cycling, instead of apoptosis, caused the reduction in cell counts, carboxyfluorescein diacetate succinimidyl diester (CFSE) staining studies were performed during the first and second weeks of culture. CFSE is an intracellular fluorescent stain, the intensity of which halves with each cell division. Three or four fewer erythroblast cell divisions were detected in 30uM DFO during the 14-day culture period when compared to controls. Based upon the reduction in cell divisions, it was estimated that the average cell cycle time was prolonged by 40–70% in DFO. Propidium iodide analyses additionally demonstrated changes in the cell cycle kinetics including a significant reduction in the percentage of S-phase erythroblasts at the peak phase of proliferation (30uM DFO: 37.0 ± 1.8% vs. control: 48.7 ± 3.4%, p = 0.0063). These results suggest that iron depletion inhibits the overall growth of erythroblasts by prolonging the cell cycle rather than causing apoptosis or maturation arrest. Due to the large amount of iron required for hemoglobin production, slower cell cycles may facilitate effective erythropoiesis by allowing extra time for uptake when extracellular iron is scarce.

Description: Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Description: Key Points LIN28B regulates HbF expression in erythroblasts that are cultured from umbilical cord and adult human blood. LIN28B expression manifested a more fetal-like phenotype among adult human erythroblasts.

Description: Abstract 827 The highly-conserved Lin28 genes regulate cellular metabolism as well as the timing of developmental events and cell fates in multicellular organisms. Lin28 protein acts primarily by negatively regulating biogenesis of let-7 RNA, a microRNA family whose targets include growth-related signaling and transcription factor proteins. Published studies showed significantly increased expression of let-7 in purified adult blood reticulocytes compared to umbilical cord blood reticulocytes (1). This pattern correlates inversely with Lin28B expression. While present in the fetal liver and umbilical cord blood, Lin28B decreased to undetectable levels in adult bone marrow (2). Based upon the association of human ontogeny with hemoglobin switching, Lin28 was explored to identify novel mechanisms for hemoglobin regulation that may be useful for therapeutic application among patients with thalassemia or other hemoglobinopathies. To study the effects of Lin28B upon erythropoiesis and hemoglobin, ectopic expression of Lin28B was accomplished using retroviral transduction of human CD34+ cells cultivated ex vivo in erythropoietin-supplemented, serum-free cultures for 21 days. All experiments were performed in triplicate using cells from three separate adult volunteers. Lin28B over-expression (Lin28B-OE) was confirmed by Q-RT-PCR (control: 0.14 ± 0.37 copies/ng, Lin28B-OE: 1.8E+04 ± 353.8 copies/ng, p=0.01). Western analyses confirmed protein expression, and confocal microscopy revealed Lin28B predominantly in the cytoplasm of the transduced cells. Proliferation, maturation and morphology assays revealed that Lin28B-OE did not inhibit erythropoiesis when compared to control (empty vector) transductions. Terminal maturation with loss of CD71 from the erythroblast surface and enucleation by culture day 21 was detected in the control and Lin28B-OE samples. Expression levels of globin genes were evaluated upon Lin28B-OE by Q-RT-PCR. Lin28B-OE enhances gamma-globin mRNA expression (control: 5.14E+06 ± 2.6E+06 copies/ng, Lin28B-OE: 1.81E+07 ± 5.82E+06 copies/ng, p=0.038). Protein analysis confirmed the increased expression of gamma-globin. Fetal hemoglobin (HbF) levels were also increased in the Lin28B-OE cultures (control: 5.82 ± 4.54%, Lin28B-OE: 33.63 ± 9.38%; p=0.011). The increased HbF expression was maintained throughout differentiation including enucleated populations of culture-generated erythrocytes. Possible mechanism(s) for the increased expression of HbF caused by Lin28B-OE were investigated. Q-RT-PCR analyses demonstrated suppression of the let-7 microRNA family with greater-than 70% reductions of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. Expression patterns of several transcription factors including BCL11A, KLF1, SOX6 and GATA1 were explored. No major changes were detected with the exception of BCL11A. Lin28B-OE caused a 65% reduction in BCL11A expression (control: 3.07E+03 ± 1.5E+02 copies/ng, Lin28B-OE: 1.07E+03 ± 18 copies/ng; p=0.02). Western blot analyses of Lin28B-OE showed a consistent reduction of BCL11A protein. By comparison with Lin28B-OE, separately performed studies of BCL11A knockdown in adult CD34+ cells produced comparable increases in gamma-globin expression, but Lin28B expression in those cells was not affected. In addition to a more general role in development and metabolism, these experimental results suggest that Lin28B increases fetal hemoglobin and regulates BCL11A in human erythroblasts. Lin28B is thus identified as the first defined link between the regulation of a developmental clock and hemoglobin switching in humans. Disclosures: No relevant conflicts of interest to declare.

Description: In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.