ALBERT

Description: Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.

Description: Iron overload and bony abnormalities cause considerable morbidity among patients with thalassemia syndromes. One possible explanation for this phenomenon is that proteins normally secreted into the marrow microenvironment during erythropoiesis are over-expressed in thalassemia patients due to expanded and ineffective erythropoiesis. We previously discovered that GDF15 is produced at very high levels in thalassemia patients and inhibits hepcidin expression. Transcriptome screens of erythroblasts were utilized here to identify twisted gastrulation (TWSG1) as a second candidate protein for further study. Quantitative PCR using the β-thalassemia murine model (Hbbth3/+ β-thalassemia intermedia mouse model, n=13; Hbbth3/th3 β-thalassemia major mouse model, n=5) revealed that splenic expression levels of Tsg (murine TWSG1) were significantly higher in thalassemia mice (Hbbth3/+, 2.2E02 ± 2.7E01 copies/ng RNA, p

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: 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.