BACH1 regulates erythrophagocytosis and iron-recycling in ß-thalassemia.

Macrophages play essential roles in erythrophagocytosis and iron recycling. ß-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of ß-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in ß-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with ß-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.

Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis.

Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.

Upregulation of miR-214 Mediates Oxidative Stress in Hb H Disease via Targeting of ATF4.

Thalassemia is a genetic disorder, occurring because of an imbalance in the globin chain production. Oxidative stress in erythroid cells of thalassemia is mainly generated from excess globin chains, by Fenton reaction, leading to hemolysis and ineffective erythropoiesis. Previously, data has shown that microRNAs (miRNAs) are involved in oxidative stress regulation in red blood cells (RBCs). microR-214 has been reported to respond with an external oxidative stress in erythroid cells by modulating activating transcription factor 4 (ATF4). In this study, we illustrated the expressions of miR-214 and ATF4 in Hb H (ß4) disease, and Hb E (HBB: c.79G>A)/ß-thalassemia (ß-thal) reticulocyte samples. Our results showed miR-214 expression was increased in Hb H disease, but not significantly different in Hb E/ß-thal reticulocytes. The ATF4 target was decreased in both thalassemic groups. Moreover, miR-214 expression level positively correlated with the reactive oxygen species (ROS) level, while it was negatively correlated with mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH) and mean corpuscular Hb concentration (MCHC). We suggested that the upregulation of miR-214 correlated with the oxidative stress as well as anemia severity of Hb H disease patients, by suppression of ATF4. Understanding the oxidative pathways in erythrocyte could be useful to manage and relieve the clinical manifestation, such as anemia, in thalassemic patients.

Anemia Severity in ß-Thalassemia Correlates with Elevated Levels of microRNA-125b in Activated Phagocytic Monocytes.

ß-Thalassemia (ß-thal), is an inherited blood disorder caused by reduced or absent synthesis of ß-globin chains leading to imbalance of globin chain synthesis. The clearance of ß-thalassemic abnormal red blood cells (RBCs) that result from excessive unbound α-globin is mainly achieved by activated monocytes. The phagocytic activity of ß-thal monocytes significantly increases when co-cultured with normal and ß-thal RBC individuals compare to that of normal monocytes co-cultured with normal RBCs. The present study indicates that microRNA (miR) plays a role in monocyte activation. In this study, we identified the higher miR-125b expression in CD14 marker-positive monocytic cells of ß-thal patients. Moreover, miR-125b expression levels positively correlate with the phagocytic activity of monocytes. Remarkably, miR-125b expression levels are negatively correlated with RBC count, hemoglobin (Hb) and hematocrit [or packed cell volume (PCV)], which are the indices for the severity of anemia. From these findings, our future studies will be to prove the hypothesis that miR-125b expression in activated monocytes may be a genetic modifier related to the severity of anemia in ß-thal patients.

Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis.

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.

Expression of microRNA-155 in thalassemic erythropoiesis.

Background: Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in ß-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with ß-thalassemia/HbE. However, the expression and function of miR-155, especially in ß-thalassemia, have not yet been explored. Methods: To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from ß IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. Results: miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in ß-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in ß-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in ß-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. Conclusion: miR-155 regulates IE in ß-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and ß-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in ß- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in ß- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

Molecular epidemiology and hematological profiles of hemoglobin variants in southern Thailand.

Data on hemoglobin (Hb) variants in southern Thailand are lacking. This study aimed to reassess the frequency of Hb variants and the clinical aspects of compound heterozygous Hb variant with other hemoglobinopathies. We enrolled 13,391 participants from ten provinces in southern Thailand during 2015-2022. Hb analysis was performed using capillary electrophoresis, and mutations in the HBA and HBB genes were identified using PCR or DNA sequencing. Hb variants were identified in 337 (2.5%) unrelated subjects. Nine ß-chain variants, namely Hb Malay (76.9%), Hb C (10.1%), Hb D-Punjab (2.9%), Hb G-Makassar (2.3%), Hb Dhonburi (2.3%), Hb Tak (1.4%), Hb J-Bangkok (1.4%), Hb New York (0.3%), and Hb Hope (0.3%), and four α-chain variants-Hb G-Georgia (HBA1) (0.9%), Hb G-Georgia (HBA2) (0.3%), Hb Q-Thailand (0.6%), and Hb St. Luke's-Thailand (0.3%)-were identified. The southern population exhibited a distinct spectrum of Hb variants compared to that observed in the populations from other areas. Several compound heterozygous genotypes were also identified. Combining Hb Malay with Hb E or high Hb F determinants did not require a blood transfusion. This study provides essential information for genetic counseling in thalassemia prevention and control programs in this region.

Reduced ADAMTS13 activity and high D-dimer levels are associated with thrombosis in patients with systemic lupus erythematosus.

INTRODUCTION: Patients with systemic lupus erythematosus (SLE) have an increased risk of vascular thrombosis compared to the general population. Therefore, biomarkers for predicting the risk of thrombosis in patients with SLE are needed. METHODS: In the present study, a total of 66 patients with SLE (22 with and 44 without a history of thrombosis) were enrolled. The cases with thrombosis and the controls without thrombosis were matched for age (± 5 years) and sex. We assessed ADAMTS13 activity, D-dimer levels, and antiphospholipid antibodies. Clinical manifestations, SLE disease activity, classical risk factors, and medical history were collected. RESULTS: ADAMTS13 activity was significantly reduced, and D-dimer levels were significantly increased in patients with SLE with a history of thrombosis compared with those in patients without thrombosis. Receiver operating characteristic curve analysis revealed a good correlation between reduced ADAMTS13 activity and a history of thrombosis. Reduced ADAMTS13 activity was correlated with increased D-dimer levels only in the thrombotic group. CONCLUSION: Reduced ADAMTS13 activity and high D-dimer levels are associated with thrombosis and may serve as prognostic markers for thrombosis in patients with SLE.

BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7.

Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

Expression of microRNA-451 in normal and thalassemic erythropoiesis.

MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis. Thalassemia, a defective globin synthesis leading to precipitate of excess unbound globins in red blood cell precursors, results in defective erythroid precursors and ineffective erythropoiesis. Expression pattern of miR-451, an erythroid-specific miRNA, was analyzed during differentiation of erythroid progenitors derived from normal and thalassemic peripheral blood CD34-positive cells, after 14 days of culture. A biphasic expression with transient up-regulation of miRNA-451 on day 3 of cultures was observed during thalassemic erythroid differentiation. In contrast, the expression pattern of the miR-451 in erythroid cells obtained from the other extravascular hemolytic anemia, i.e., hereditary spherocytosis patients showed no transient up-regulation of miR-451 on day 3 of cultures. Our results suggest that early erythroid progenitors in beta-thalassemia have a dysregulated miRNA-451 expression program, and analysis of microRNA is a relevant approach to determine abnormalities of erythropoiesis.

The effects of Phorbol 12-myristate 13-acetate concentration on the expression of miR-155 and miR-125b and their macrophage function-related genes in the U937 cell line.

The phorbol 12-myristate 13-acetate (PMA)-induced U937 cell line has been widely used as an in vitro model for studying the functions of human macrophages. However, there are several concentrations of PMA commonly used to drive the differentiation of monocytic cell line to macrophage. Also, the expression of microRNA-155 (miR-155) and miR-125b in PMA-treated human monocytic cell line has not yet been reported. The five usual concentrations of PMA for stimulating macrophage differentiation are 10, 25, 50, 100, and 200 nM. In this study we compared the expression levels of miR-155, miR-125b and their related genes involved in macrophage functions in U937-derived cells after treatment with those five concentrations. The morphological study results showed that the five concentrations of PMA could induce macrophage differentiation in a similar manner. Moreover, cell proliferation and viability were not significantly different among these five conditions excepted the lower cell viability at 200 nM of PMA treatment. The five concentrations of PMA could upregulate the expression of miR-155 and miR-125b and increase the phagocytic activity of U937-derived cells in dose-reversal manner. The upregulation of miR-155 was correlated with increased expression levels of TNFα and decreased expression levels of BACH1 and CEBPß, while the reduction of IRF4 was correlated with increased expression levels of miR-125b. Our study found that PMA could stimulate macrophage differentiation in a broad range of concentrations, however, the lower concentration could upregulate the higher expression of both miR-155 and miR-125b, and that correlated with the phagocytic functional activity of U937-derived macrophages.

The LXCXE Retinoblastoma Protein-Binding Motif of FOG-2 Regulates Adipogenesis.

GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. Mammals have six GATA and two FOG factors. We recently demonstrated that interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation. We show here that the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Unlike GATA-1, which inhibits cell division, FOG-2 promotes proliferation. Mice with a knockin of a Fog2 gene bearing a mutated LXCXE pRb-binding site are resistant to obesity and display higher rates of white-to-brown fat conversion. Thus, each component of the GATA/FOG complex (GATA-1 and FOG-2) is involved in pRb/E2F regulation, but these molecules have markedly different roles in the control of tissue homeostasis.

Clinical severity of ß-thalassaemia/Hb E disease is associated with differential activities of the calpain-calpastatin proteolytic system.

Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in ß-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in ß-thalassaemia. CD34(+) cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of ß(0)-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of µ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms.