Abstract
Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of α-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.
Highlights
The overall view of erythropoiesis remains unclear
The concentrations of each chemical were reduced for comparable microarray analysis, because general concentrations, 1.5% dimethyl sulfoxide (DMSO), 5.0 mM hexamethylene bisacetamide (HMBA), or 30 nM trichostatin A (TSA), were affecting the growth of low differentiation-inducible (LD) murine erythroleukemia (MEL) cells
We compared the expression profiles of high differentiation-inducible (HD) and LD MEL cells during differentiation induced by 1.0% DMSO, 3.0 mM HMBA, or 15 nM TSA using a cDNA microarray that was prepared from NIA mouse 15k and 7.4k cDNA clone sets
Summary
The overall view of erythropoiesis remains unclear. Results: Overexpression of ␣-1,6-fucosyltransferase inhibits hemoglobin production in murine and human erythroleukemia cells; down-regulation of ␣-1,6-fucosyltransferase promotes hemoglobin production and erythroid differentiation of human erythroleukemia cells. A short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. FUT8 Inhibits Hb Production in MEL and K562 Differentiation regulated by Epo stimulation and by transcriptional control with the development-specific transcription factor GATA-1. Both FLI-1 and EKLF/KLF1 bind to GATA-1 and are functionally antagonistic to the activation of megakaryocytic and erythrocytic gene promoters [20] They control megakaryocytic and erythrocytic differentiation of MEPs. A recent study showed that microRNA-145 (miR-145) represses Fli-1 [21]. The addition of chemicals, such as dimethyl sulfoxide (DMSO), hexamethylene bisacetamide (HMBA), and trichostatin A (TSA), is known to induce differentiation of MEL cells to erythroblasts that highly express hemoglobin [23,24,25] These chemicals act as initiators of the synthesis of -globin and other erythroid-specific proteins [24]. We performed functional analysis of FUT8 to understand the mechanism of suppression of hemoglobin production and erythroid differentiation, as observed in MEL and K562 cells
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