Y. Terry Lee

High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin

In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 ± 50 pg/ml. In comparison, individuals with β-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 ± 9,600 pg/ml; range 4,800–248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.

Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells

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.

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo

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.

Let-7 microRNAs are developmentally regulated in circulating human erythroid cells

BackgroundMicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching.MethodsExpression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample.ResultsAmong 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells.ConclusionThese data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.

Cloning and Characterization of a Gene Expressed during Terminal Differentiation That Encodes a Novel Inhibitor of Growth

We report here the cloning and initial characterization of a novel growth-related gene (EEG-1) that is located on the short arm of chromosome 12. Two spliced transcripts were cloned from human bone marrow and human erythroid progenitor cells: EEG-1L containing a 4350-nucleotide open reading frame encoding a putative protein of 1077 amino acids including a C1q-like globular domain, and an alternatively spliced transcript lacking exon 5 (EEG-1S) encodes a significantly smaller coding region and no C1q-like domain. Quantitative PCR revealed expression of both EEG-1 transcripts in all analyzed tissues. Plasmids encoding green fluorescent protein-tagged genes (GFP-EEG-1) were transfected into Chinese hamster ovary cells for localization and functional assays. In contrast to the diffuse cellular localization of the GFP control, GFP-EEG-1L was detected throughout the cytoplasm and excluded from the nucleus, and GFP-EEG-1S co-localized with aggregated mitochondria. Transfection of both isoforms was associated with significantly increased levels of apoptosis. Stable transfection assays additionally demonstrated decreased growth in those cells expressing EEG-1 at higher levels. Quantitative PCR analyses of mRNA obtained from differentiating erythroid cells from blood donors were performed to determine the transcriptional pattern of EEG-1 during erythropoiesis. EEG-1 expression was highly regulated with increased expression at the stage of differentiation associated with the onset of global nuclear condensation and reduced cell proliferation. We propose that the regulated expression of EEG-1 is involved in the orchestrated regulation of growth that occurs as erythroblasts shift from a highly proliferative state toward their terminal phase of differentiation.

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping

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.

Expression of growth differentiation factor 15 is not elevated in individuals with iron deficiency secondary to volunteer blood donation.

BACKGROUND: Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.

Hembase: browser and genome portal for hematology and erythroid biology

Hembase (http://hembase.niddk.nih.gov) is an integrated browser and genome portal designed for web-based examination of the human erythroid transcriptome. To date, Hembase contains 15,752 entries from erythroblast Expressed Sequenced Tags (ESTs) and 380 referenced genes relevant for erythropoiesis. The database is organized to provide a cytogenetic band position, a unique name as well as a concise annotation for each entry. Search queries may be performed by name, keyword or cytogenetic location. Search results are linked to primary sequence data and three major human genome browsers for access to information considered current at the time of each search. Hembase provides interested scientists and clinical hematologists with a genome-based approach toward the study of erythroid biology.

HEPCIDIN, ANAEMIA, AND PROSTATE CANCER

Sir, Anaemia is a common haematological abnormality in patients with cancer. The anaemia of chronic disease or inflammation is thought to be a result of induction of the iron-regulatory hormone, hepcidin, by inflammatory cytokines, especially interleukin 6 (IL-6) [1]. Elevation of hepcidin by IL-6 leads to hypoferraemia associated with anaemia and iron-restricted erythropoiesis [2]. A recent study showed that patients with certain early-stage malignancies are less prone to developing anaemia of chronic disease [3]. Among more than 1400 men with prostate cancer, the odds of developing anaemia before diagnosis were not increased. We hypothesized that the paucity of significant anaemia during the early stages of prostate cancer may be due to potentially balanced effects of hepcidin-regulating cytokines produced by prostate cancer cells. Like IL-6, growth differentiation factor 15 (GDF15), also a cytokine, is overexpressed in prostate cancer tissues [4,5]. However, unlike IL-6, GDF15 suppresses hepcidin expression [6]. Hepcidin, IL-6 and GDF15, as well as iron and haematological variables were therefore examined to determine the potential role of iron-restricted anaemia in men with prostate cancer. In all, 29 men with prostate cancer were recruited for this preliminary study and grouped according to the presence or absence of metastatic bone disease. All studies were conducted at the National Institutes of Health (NIH) and were approved by both the Office of Human Subjects Research and the Institutional Review Board. Mild anaemia (defined here as a haemoglobin [Hb] level of 10.0–13.7 g/dL) was detected in >80% of men in both groups (Fig. 1). As expected, Hb levels were lower in men with metastatic bone disease, with a mean (SD) Hb of 11.6 (1.4) g/dL (P = 0.009) and haematocrit (Hct) of 36.5 (4.1)% (P = 0.025), than in men without metastasis, with a mean (SD) Hb of 12.8 (0.8) g/dL and Hct of 39.4 (2.1)%. None of the 29 men had received erythropoietin or transfused blood, and only one had received iron supplements within the 6 weeks prior to the study. The iron parameters were within the normal range in >80% of the men. FIG. 1 Hepcidin, IL-6, and GDF15 levels in men with prostate cancer with or with no bone metastasis. Serum concentrations of (A) hepcidin, (B) IL-6, and (C) GDF15 from men with prostate cancer, without (−, 14 men) or with (+, 15 men) bone metastasis. ... ELISA-based assay systems were used to examine the serum levels of hepcidin, GDF15 and IL-6 [6, 7]. As shown in the attached Figure 1A, 23 of 29 men studied had measurable hepcidin within the assay’s normal range (10–298 ng/mL) [7]. Five men had hepcidin levels at or near the lower limit of the assay (5.0 ng/mL). Serum hepcidin level increased beyond the normal range only in one man. In the absence of bone metastasis, hepcidin levels clustered in the low normal range, with a mean value of 42 ng/mL. Even with bone metastasis, over half of the men maintained hepcidin levels of <100 ng/mL, but values were more broadly distributed within the normal range. Statistical analyses showed that the mean (SD) hepcidin level was significantly increased, at 121 (111) ng/mL (P = 0.028) in men with bone metastasis compared with men with no metastasis, at 42.0 (64.2) ng/mL. Further comparison showed that IL-6 levels increased in men with bone metastasis (Fig. 1B), while GDF15 levels increased in both groups. However, increases in GDF15 were more robust and consistent in the men with metastatic disease (Fig. 1C). These data support the notion that hepcidin regulation, and its contribution to subsequent development of anaemia, is complex in men with prostate cancer. In addition to IL-6, serum GDF15 may play a role in hepcidin regulation. Increased serum GDF15 is associated with disease progression in men with prostate cancer, and expression of GDF15 is significantly higher in cancerous prostatic tissue than in normal tissue [4, 5]. Larger studies are needed to determine if the high level of GDF15 combined with mild elevation of serum IL-6 have balanced effects upon serum hepcidin levels in men with prostate cancer.

Increased Reticulocytosis during Infancy Is Associated with Increased Hospitalizations in Sickle Cell Anemia Patients during the First Three Years of Life

Objective Among older children with sickle cell anemia, leukocyte counts, hemoglobin, and reticulocytosis have previously been suggested as disease severity markers. Here we explored whether these blood parameters may be useful to predict early childhood disease severity when tested in early infancy, defined as postnatal ages 60–180 days. Study Design Data from fifty-nine subjects who were followed at Children’s National Medical Center’s Sickle Cell Program for at least three years was retrospectively analyzed. Comparisons were made between white blood cell counts, hemoglobin and reticulocyte levels measured at ages 60–180 days and the clinical course of sickle cell anemia during infancy and childhood. Results A majority of subjects had demonstrable anemia with increased reticulocytosis. Only increased absolute reticulocyte levels during early infancy were associated with a significant increase in hospitalization during the first three years of life. Higher absolute reticulocyte counts were also associated with a markedly shorter time to first hospitalizations and a four-fold higher cumulative frequency of clinical manifestations over the first three years of life. No significant increase in white blood cell counts was identified among the infant subjects. Conclusions These data suggest that during early infancy, increased reticulocytosis among asymptomatic SCA subjects is associated with increased severity of disease in childhood.