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The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2., (© 2016 by The American Society of Hematology.)

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies.

Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.

The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the (A)γ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

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.

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.

Congenital dyserythropoietic anemia (CDA) is a rare group of red blood cell disorders characterized by ineffective erythropoiesis and increased iron absorption. To determine whether growth differentation factor 15 (GDF15) hyper-expression is associated with the ineffective erythropoiesis and iron-loading complications of CDA type I (CDA I), GDF15 levels and other markers of erythropoiesis and iron overload were studied in blood from 17 CDA I patients. Significantly higher levels of GDF15 were detected among the CDA I patients (10 239 +/- 3049 pg/mL) compared with healthy volunteers (269 +/- 238 pg/mL). In addition, GDF15 correlated significantly with several erythropoietic and iron parameters including Hepcidin-25, Ferritin, and Hepcidin-25/Ferritin ratios. These novel results suggest that CDA I patients express very high levels of serum GDF15, and that GDF15 contributes to the inappropriate suppression of hepcidin with subsequent secondary hemochromatosis.

Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a "switching" model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

A previously undefined transcript with significant homology to the pseudo-alpha2 region of the alpha-globin locus on human chromosome 16 was detected as part of an effort to better define the transcriptional profiles of human reticulocytes. Cloning and sequencing of that transcript (GenBank AY698022; named mu-globin) revealed an insert with a 423-nucleotide open reading frame. BLASTP and ClustalW and phylogenetic analyses of the predicted protein demonstrated a high level of homology with the avian alpha-D globin. In addition, the heme- and globin-binding amino acids of mu-globin and avian alpha-D globin are largely conserved. Using quantitative real-time polymerase chain reaction (PCR), mu-globin was detected at a level of approximately 0.1% that measured for alpha-globin in erythroid tissues. Erythroid-specific expression was detected by Northern blot analysis, and maximal expression during the erythroblast terminal differentiation was also detected. Despite this highly regulated pattern of mu-globin gene transcription, mu-globin protein was not detected by mass spectrometry. These results suggest the human genome encodes a previously unrecognized globin member of the avian alpha-D family that is transcribed in a highly regulated pattern in erythroid cells.

We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factor beta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 +/- 1.0%; EPO + TGF-B = 10.8 +/- 1.9%; EPO + SCF = 19.1 +/- 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 +/- 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 +/- 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 +/- 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation.

Increases in fetal hemoglobin have been identified after birth in several clinical settings associated with stressed or malignant erythropoiesis. To better understand the relationship between the expression of this fetal protein and growth, donated human erythroid progenitor cells were cultured in the presence of erythropoietin (EPO) plus the growth-modifying cytokine stem cell factor (SCF), and several growth-related signaling pathways were interrogated. Only the MEK1/2 inhibitor (PD98059) demonstrated significant effects on fetal hemoglobin. In the absence of PD98059, levels of fetal hemoglobin averaged 27.4% +/- 7.9% in EPO+SCF compared with 1.26% +/- 1.7% in EPO alone (P =.02). A linear dose response in levels of fetal hemoglobin to PD98059 was detected (0.16 microM = 27.13%, 0.8 microM = 19.6%, 4 microM = 12.2%, 20 microM = 1.54%). Western blot analyses revealed that SCF was required for phosphorylation of MEK and p44MAPK in this setting, and quantitative polymerase chain reaction demonstrated a significant increase in gamma-globin mRNA. Particular perturbations of growth-related signaling may also function to activate tissue-specific genes normally expressed during fetal development. This concept may be relevant for the development of new treatment rationales for beta hemoglobinopathies.

Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)-based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

Platelet-type von Willebrand disease (PTVWD) is a bleeding disorder in which an increase of function mutation in glycoprotein Ibalpha (GPIbalpha), with respect to binding of von Willebrand factor (VWF), results in a loss of circulating high molecular weight VWF multimers together with a mild-moderate thrombocytopenia. To better ascertain the specific perturbations in adhesion associated with this disease state, we performed a detailed analysis of the kinetic and mechanical properties of tether bonds formed between PT-VWD platelets and the A1-domain of VWF. Results indicate that the GPIbalpha mutation, Gly233Val, promotes and stabilizes platelet adhesion to VWF at shear rates that do not support binding between the native receptor-ligand pair due to enhanced formation and increased longevity of the mutant tether bond (k0 off values for mutant versus native complex of 0.67 +/- 0.11 s-1 and 3.45 +/- 0.37 s-1, respectively). By contrast, the sensitivity of this interaction to an applied force, a measure of bond strength, was similar to the wild-type (WT) receptor. Although the observed alterations in the intrinsic properties of the GPIbalpha-VWF tether bond are comparable to those reported for the type 2B VWD, distinct molecular mechanisms may be responsible for these function-enhancing bleeding disorders, as interactions between the mutant receptor and mutant ligand resulted in a greater stability in platelet adhesion. We speculate that the enhanced cellular on-rate together with the prolongation in the lifetime of the mutant receptor-ligand bond contributes to platelet aggregation in circulating blood by permitting the formation of multiple GPIbalpha-VWF-A1 interactions.

A cytokine-screening assay of cultured peripheral blood cells obtained using immune rosetting and separation of progenitors was developed to identify determinants of fetal hemoglobin (HbF) modulation during adult erythropoiesis. Among the 12 erythroid growth-promoting cytokines tested, stem cell factor (SCF) at a concentration of 50 ng/mL resulted in the most significant increase in cell proliferation and HbF content. The average HbF/hemoglobin A (HbA) ratio was 30.9% +/- 18.7% in cultures containing SCF compared with 4.1% +/- 2.2% in those grown with erythropoietin (EPO) alone (P = 8.5E-8). To further investigate the hemoglobin-modulating effects of SCF, we examined the surface expression pattern of the SCF receptor, CD117, among maturing erythroblasts. CD117 expression increased during the first week of culture and peaked on culture days 7 to 9. After culture day 9, the level of CD117 declined to lower levels. The rise in CD117 expression to high levels mirrored that of the transferrin receptor (CD71), and the subsequent reduction in CD117 was inversely related to increases in expression of glycophorin A. SCF-related increases in the HbF/HbA ratio correlated with the expression pattern of CD117. SCF added during days 7 to 14 resulted in a more pancellular distribution of HbF on day 14 compared with the heterocellular distribution present in cultures supplemented with SCF on days 0 to 7. A significant SCF-mediated increase in HbF was also measured using progenitors derived from cord blood. These results suggest that the HbF response to SCF is greatest at the late progenitor stage as a function of surface CD117 expression.

The design and evaluation of therapies for the sickle cell and beta-thalassemia syndromes rely on our understanding of hemoglobin accumulation during human erythropoiesis. Here we report direct measurements of hemoglobin composition and messenger RNA (mRNA) levels in cultured CD34(+) cells and correlate those measurements with studies of freshly obtained bone marrow samples. Hemoglobin levels in differentiating cells were also compared with morphologic, immunophenotypic, and cell cycle assessments. A population of large preproerythroblasts was first identified within 24 hours and became the dominant population by day 5. The transition from proerythroblast to basophilic normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1% +/- 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts were the dominant and final cell type by day 13. High-performance liquid chromatography-based quantitation of fetal (HbF) and adult (HbA) hemoglobin and real-time polymerase chain reaction globin mRNA quantitation demonstrated a coordinate rise in the accumulation of both proteins and mRNA among these developmentally staged populations. Quantitative analyses on freshly sorted bone marrow populations demonstrated a similar rising pattern with beta-globin and HbA as the dominant species at both early and late stages of differentiation. We found no evidence for HbF dominant populations or switching during differentiation in adult cells. Instead, rapid increases in both HbF (heterocellular) and HbA (pancellular) content were observed, which coincided with the apex in cell cycling and the proerythroblast-basophilic normoblast transition. Based on these measurements, we conclude that HbF and HbA content are regulated with the rate of proliferation during adult erythropoiesis.

Because abnormal erythroid differentiation is the most common manifestation of the myelodysplastic syndromes (MDS), it was hypothesized that erythroid gene expression may be used to illustrate myelodysplastic transcription patterns. Ten normal bone marrow aspirates (NBM) were first analyzed using an erythroid-focused cDNA array to define steady-state transcription levels. Proliferation and differentiation gene subsets were identified by statistically significant differences between NBM and erythroleukemia gene expression. Next, cDNAs from 5 separate MDS aspirates were studied: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation (RAEB-T), and RAEB-T/secondary MDS. A distinct pattern of significantly increased proliferation-associated and reduced differentiation-associated gene activity was established for MDS.

Human porphobilinogen deaminase (PBGD) is, reportedly, encoded by 2 distinct messenger RNAs (mRNAs) transcribing from a single gene. The ubiquitous form of the PBGD gene product is often used as an endogenous reference in gene expression studies because it is pseudogene free and has minimal transcriptional variability among tissues. A distinct erythroid-specific gene product has also been described because of the alternate splicing of the gene. Here is reported the existence of an additional erythroid-specific isoform of PBGD mRNA in primary cells.

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the "Dombrock" blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5'-diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Do(a) versus Do(b) antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.