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 BMPmediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes. IntroductionSystemic iron homeostasis in mammals is largely maintained by the effects of hepcidin, 1 a small protein produced by hepatocytes. Hepcidin is regulated at the transcriptional and posttranscriptional levels by multiple extracellular signals related to iron homeostasis and inflammation. Erythropoiesis is also thought to regulate hepcidin expression through a variety of mechanisms including anemia-related hypoxia and erythropoietin production. ␤-Thalassemia syndromes are congenital anemias caused by mutations that reduce or abolish ␤-globin gene expression. Despite the common feature of decreased globin chain synthesis in all patients, there are prominent phenotypic variations in the disease that are not fully understood. 2 In so-called "iron-loading" anemias like thalassemia, the diseased erythron dysregulates iron homeostasis by inhibiting hepcidin expression even in the presence of severe iron overload. Humans with thalassemia syndromes express very high levels of a cytokine named GDF15, and GDF15 present in thalassemia patients' sera inhibited hepatic hepcidin expression ex vivo. 3 However, thalassemia sera also suppressed hepcidin expression to a lesser degree after immunoprecipitation of GDF15. 3 It was therefore hypothesized that GDF15 may act with other molecules to suppress hepcidin.In addition to clinical research in humans, murine models were developed for studies of thalassemia and hepcidin regulation. Mice with deletions of both the ␤ minor and ␤ major genes (th3 genotype) have a ␤-thalassemia intermedia phenotype in the heterozygous state. The homozygous deletion (th3/th3) results in death...

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.Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and b-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-toadult 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 CD341 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 g-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-toadult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of g-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. (Blood. 2013;122(6):1034-1041 IntroductionIn humans and some other mammals, the composition of hemoglobin tetramers in erythrocytes switch from fetal hemoglobin (HbF) (a2g2) to adult hemoglobin (HbA) (a2b2) during the last stages of fetal development until early infancy.1 HbF is the most important known modifier of the clinical symptoms for patients with sickle cell disease (SCD) and b-thalassemias, which are among the most common genetic disorders worldwide. 2,3 In patients with SCD, the polymerization of sickle hemoglobin results in erythrocyte deformation and hemolysis.4 SCD patient's clinical outcomes are largely improved by inhibition of the polymerization by HbF. 5 In b-thalassemias, decreased production of b-globin causes imbalanced globin polypeptide chain synthesis, and leads to severe effects on the erythroid cells' maturation and survival. The loss of b-globin expression may be compensated by an increase in HbF production that leads to improvement of the clinical phenotype. 6 The molecular mechanisms underlying the switch from HbF to HbA are still largely unknown. Genome-wide association studies (GWAS) in both normal individuals and patients with b-hemoglobinopathies have identified BCL11A, HSB1L-MYB, and HBB clusters as having an association with the persistence of Hb...

RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe generated from the reticulocyte total RNA (blood obtained from 14 umbilical cords and 14 healthy adult humans). Among the cord and adult reticulocyte profiles, 698 probe sets (488 named genes) were detected in each of the 28 samples. Among the highly expressed genes, promoter analyses revealed a subset of transcription factor binding motifs encoded at higher than expected frequencies including the hypoxia-related arylhydrocarbon receptor repressor family. Over 100 probe sets demonstrated differential expression between the cord and adult reticulocyte samples. For verification, the array expression patterns for 21 genes were confirmed by real-time PCR (correlation coefficient 0.98). Only four transcripts (MAP17, FLJ32009, ARRB2, and FLJ27365) were identified as being upregulated in the adult blood transcriptome. Further analysis revealed that the lipid-regulating protein MAP17 was present in the membrane fraction of adult erythrocytes, but not detected in cord blood erythrocytes. Combined with other clinical and experimental data, these reticulocyte transcriptome profiles should be useful to better understand the molecular bases of terminal erythroid differentiation, hemoglobin switching, iron metabolism and malarial pathogenesis.

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