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α-1,6-Fucosyltransferase (FUT8) Inhibits Hemoglobin Production during Differentiation of Murine and K562 Human Erythroleukemia Cells*

  • Hitoshi Sasaki
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Takanori Toda
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Toru Furukawa
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Yuki Mawatari
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Rika Takaesu
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Masashi Shimizu
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Ryohei Wada
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101
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  • Dai Kato
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585
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  • Takahiko Utsugi
    Affiliations
    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101

    Bio Matrix Research Inc., Chiba 270-0101, Japan
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  • Masaya Ohtsu
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585
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  • Yasufumi Murakami
    Correspondence
    To whom correspondence should be addressed: Dept. of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 3-1, Niijyuku 6-chome, Katsushika-ku, Tokyo 125-8585, Japan , Tel.: 81-3-5876-1717 (Ext. 1918); Fax: 81-3-5875-1470;
    Affiliations
    Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585

    Genome and Drug Research Center, Tokyo University of Science, Chiba 270-0101

    Bio Matrix Research Inc., Chiba 270-0101, Japan
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Bio Matrix Research Inc.
    This article contains supplemental material.
Open AccessPublished:April 22, 2013DOI:https://doi.org/10.1074/jbc.M113.459594
      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.
      Background: The overall view of erythropoiesis remains unclear.
      Results: Overexpression of α-1,6-fucosyltransferase inhibits hemoglobin production in murine and human erythroleukemia cells; down-regulation of α-1,6-fucosyltransferase promotes hemoglobin production and erythroid differentiation of human erythroleukemia cells.
      Conclusion: Core fucosylation plays an important role in hemoglobin production and erythroid differentiation.
      Significance: This might be the first finding that glycosylation negatively regulates erythroid differentiation.

      Introduction

      Erythropoiesis is intricately regulated by several linage-specific factors (
      • Orkin S.H.
      • Zon L.I.
      Hematopoiesis: an evolving paradigm for stem cell biology.
      ). Pro-erythroblasts/colony-forming unit-erythroid cells, which arise from megakaryocytic/erythroid progenitors (MEPs),
      The abbreviations used are: MEP, megakaryocytic/erythroid progenitor; FUT8, α-1,6-fucosyltransferase; MEL, murine erythroleukemia; Epo, erythropoietin; EpoR, erythropoietin receptor; HMBA, hexamethylene bisacetamide; TSA, trichostatin A; HD, high differentiation-inducible; LD, low differentiation-inducible; RRM, R365A/R366A double mutant; FLM, D368A/K369A/V370A/G371A/T372A quintuple mutant; TGF-β, transforming growth factor β1; SH, Src homology.
      are stimulated by erythropoietin (Epo), differentiate into erythroblasts, and finally maturate to become erythrocytes/red blood cells (
      • Wu H.
      • Liu X.
      • Jaenisch R.
      • Lodish H.F.
      Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
      ,
      • Lin C.S.
      • Lim S.K.
      • D'Agati V.
      • Costantini F.
      Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.
      ,
      • Kieran M.W.
      • Perkins A.C.
      • Orkin S.H.
      • Zon L.I.
      Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor.
      ). Binding of Epo to the Epo receptor (EpoR) leads to phosphorylation and activation of receptor-associated Janus-kinase 2 (JAK2) and initiation of the EpoR signaling cascade (
      • Witthuhn B.A.
      • Quelle F.W.
      • Silvennoinen O.
      • Yi T.
      • Tang B.
      • Miura O.
      • Ihle J.N.
      JAK2 associates with the erythropoietin receptor and is tyrosine-phosphorylated and activated following stimulation with erythropoietin.
      ,
      • Ghaffari S.
      • Kitidis C.
      • Fleming M.D.
      • Neubauer H.
      • Pfeffer K.
      • Lodish H.F.
      Erythropoiesis in the absence of Janus-kinase 2: BCR-ABL induces red cell formation in JAK2(−/−) hematopoietic progenitors.
      ). Epo also stimulates phosphorylation and activation of GATA-binding protein 1 (GATA-1) (
      • Zhao W.
      • Kitidis C.
      • Fleming M.D.
      • Lodish H.F.
      • Ghaffari S.
      Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway.
      ). The transcription factor GATA-1 is highly expressed in MEPs and is well known as a specific regulator of erythroid differentiation. GATA-1 induces the expression of erythroid genes such as glycophorin A, EpoR, and hemoglobin (
      • Weiss M.J.
      • Orkin S.H.
      Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis.
      ). In embryos of GATA-1 null mice, embryonic erythroid cells are arrested at an early pro-erythroblast-like stage of their development (
      • Fujiwara Y.
      • Browne C.P.
      • Cunniff K.
      • Goff S.C.
      • Orkin S.H.
      Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.
      ) and die thereafter by apoptosis. The transcription factor PU.1 is a hematopoietic-specific member of the ETS family that is required for the development of lymphoid and myeloid lineages (
      • Scott E.W.
      • Simon M.C.
      • Anastasi J.
      • Singh H.
      Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages.
      ). However, PU.1 interacts directly with GATA-1 and blocks terminal erythroid differentiation of murine erythroleukemia (MEL) cells by repression of GATA-1-mediated transcriptional activation (
      • Rekhtman N.
      • Radparvar F.
      • Evans T.
      • Skoultchi A.I.
      Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells.
      ). The transcription factor erythroid Kruppel-like factor (EKLF/KLF1) plays a crucial function during erythropoiesis (
      • Siatecka M.
      • Bieker J.J.
      The multifunctional role of EKLF/KLF1 during erythropoiesis.
      ). EKLF/KLF1 and KLF2 bind the c-Myc (Myc) promoter and regulate expression of the Myc gene (
      • Pang C.J.
      • Lemsaddek W.
      • Alhashem Y.N.
      • Bondzi C.
      • Redmond L.C.
      • Ah-Son N.
      • Dumur C.I.
      • Archer K.J.
      • Haar J.L.
      • Lloyd J.A.
      • Trudel M.
      Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis.
      ). Down-regulation of Myc expression at the late stage of erythropoiesis is essential for terminal erythroid maturation (
      • Jayapal S.R.
      • Lee K.L.
      • Ji P.
      • Kaldis P.
      • Lim B.
      • Lodish H.F.
      Down-regulation of Myc is essential for terminal erythroid maturation.
      ). GATA-1 represses Myc and c-Myb (Myb) expression during erythroid differentiation (
      • Rylski M.
      • Welch J.J.
      • Chen Y.Y.
      • Letting D.L.
      • Diehl J.A.
      • Chodosh L.A.
      • Blobel G.A.
      • Weiss M.J.
      GATA-1-mediated proliferation arrest during erythroid maturation.
      ,
      • Bartnek P.
      • Králová J.
      • Blendinger G.
      • Dvorák M.
      • Zenke M.
      GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter.
      ). As described above, erythropoiesis is predominantly regulated by Epo stimulation and by transcriptional control with the development-specific transcription factor GATA-1.
      Friend leukemia integration 1 (FLI-1) is one of the well known regulators of erythropoiesis. FLI-1, a member of the ETS family of transcription factors that was originally identified in MEL cells, has a role in erythroleukemia induction (
      • Ben-David Y.
      • Giddens E.B.
      • Bernstein A.
      Identification and mapping of a common proviral integration site Fli-1 in erythroleukemia cells induced by Friend murine leukemia virus.
      ). FLI-1 is also expressed in normal hematopoietic cells and suppresses erythroid differentiation (
      • Athanasiou M.
      • Mavrothalassitis G.
      • Sun-Hoffman L.
      • Blair D.G.
      FLI-1 is a suppressor of erythroid differentiation in human hematopoietic cells.
      ). However, FLI-1 activates megakaryocytic differentiation (
      • Athanasiou M.
      • Clausen P.A.
      • Mavrothalassitis G.J.
      • Zhang X.K.
      • Watson D.K.
      • Blair D.G.
      Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype.
      ). Both FLI-1 and EKLF/KLF1 bind to GATA-1 and are functionally antagonistic to the activation of megakaryocytic and erythrocytic gene promoters (
      • Starck J.
      • Cohet N.
      • Gonnet C.
      • Sarrazin S.
      • Doubeikovskaia Z.
      • Doubeikovski A.
      • Verger A.
      • Duterque-Coquillaud M.
      • Morle F.
      Functional cross-antagonism between transcription factors FLI-1 and EKLF.
      ). Moreover, they control megakaryocytic and erythrocytic differentiation of MEPs. A recent study showed that microRNA-145 (miR-145) represses Fli-1 (
      • Kumar M.S.
      • Narla A.
      • Nonami A.
      • Mullally A.
      • Dimitrova N.
      • Ball B.
      • McAuley J.R.
      • Poveromo L.
      • Kutok J.L.
      • Galili N.
      • Raza A.
      • Attar E.
      • Gilliland D.G.
      • Jacks T.
      • Ebert B.L.
      Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q− syndrome.
      ). That study also revealed the effects of miR-145 on megakaryocytic and erythroid differentiation. This finding indicates the existence of a novel differentiation regulator in addition to existing transcription factors, cytokines, and cytokine receptors. Thus, the overall view of erythroid differentiation is not yet clear.
      MEL and human erythroleukemia K562 (K562) cells are widely used for the study of erythroid differentiation. MEL cells, which were isolated from Friend virus-infected mice, provide a model system for the study of erythroblast differentiation and leukemogenesis (
      • Ney P.A.
      • D'Andrea A.D.
      Friend erythroleukemia revisited.
      ). 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 (
      • Friend C.
      • Scher W.
      • Holland J.G.
      • Sato T.
      Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide.
      ,
      • Marks P.A.
      • Sheffery M.
      • Ramsay R.
      • Ikeda K.
      • Rifkind R.A.
      Induction of transformed cells to terminal differentiation.
      ,
      • Yoshida M.
      • Nomura S.
      • Beppu T.
      Effects of trichostatins on differentiation of murine erythroleukemia cells.
      ). These chemicals act as initiators of the synthesis of β-globin and other erythroid-specific proteins (
      • Marks P.A.
      • Sheffery M.
      • Ramsay R.
      • Ikeda K.
      • Rifkind R.A.
      Induction of transformed cells to terminal differentiation.
      ). Expression of Myc and Myb genes are down-regulated during MEL cell differentiation, and overexpression of these genes blocks differentiation (
      • Kaneko-Ishino T.
      • Kume T.U.
      • Sasaki H.
      • Obinata. M.
      • Oishi M.
      Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.
      ,
      • Todokoro K.
      • Watson R.J.
      • Higo H.
      • Amanuma H.
      • Kuramochi S.
      • Yanagisawa H.
      • Ikawa Y.
      Down-regulation of c-myb gene expression is a prerequisite for erythropoietin-induced erythroid differentiation.
      ). Furthermore, DMSO-resistant MEL cell clones were isolated and used for the study of erythroid differentiation (
      • Fujita H.
      • Yamamoto M.
      • Yamagami T.
      • Hayashi N.
      • Sassa S.
      Erythroleukemia differentiation. Distinctive responses of the erythroid-specific and the nonspecific δ-aminolevulinate synthase mRNA.
      ,
      • Fukuda Y.
      • Fujita H.
      • Garbaczewski L.
      • Sassa S.
      Regulation of β-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells.
      ). K562 cells were isolated from the blood of patients with chronic myelogenous leukemia. Sodium butyrate and hemin also induce erythroid differentiation of K562 cells (
      • Lozzio C.B.
      • Lozzio B.B.
      Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome.
      ,
      • Andersson L.C.
      • Jokinen M.
      • Gahmberg C.G.
      Induction of erythroid differentiation in the human leukaemia cell line K562.
      ,
      • Rutherford T.
      • Clegg J.B.
      • Higgs D.R.
      • Jones R.W.
      • Thompson J.
      • Weatherall D.J.
      Embryonic erythroid differentiation in the human leukemic cell line K562.
      ). In a phenotypic analysis of K562 cells, the rate of formation of transferrin receptor (CD71)/glycophorin A-positive cells was increased by hemin-mediated induction of differentiation (
      • Di Pietro R.
      • di Giacomo V
      • Caravatta L.
      • Sancilio S.
      • Rana R.A.
      • Cataldi A.
      Cyclic nucleotide response element binding (CREB) protein activation is involved in K562 erythroleukemia cells differentiation.
      ). During erythroid differentiation, CD71 is highly expressed in the pre-proerythroblast and the erythroblasts that follow, whereas glycophorin A expression is delayed relative to CD71 and correlates with the transition from the pro-erythroblast to the basophilic normoblast (
      • Wojda U.
      • Noel P.
      • Miller J.L.
      Fetal and adult hemoglobin production during adult erythropoiesis: coordinate expression correlates with cell proliferation.
      ). Although MEL and K562 cells are erythroleukemia cells, these cells are useful models for the study of erythroid differentiation because they can be induced to differentiate like normal erythroid cells.
      We here used DNA microarrays to screen for erythroid differentiation-related genes to identify novel regulators of hemoglobin production and erythroid differentiation. We compared the expression profile of high differentiation-inducible (HD) and low differentiation-inducible (LD) MEL cells during differentiation induced by three different chemicals. We selected the consistently down-regulated genes in HD MEL cells as candidate differentiation suppressors and then overexpressed these genes in HD MEL cells to analyze for inhibition of hemoglobin production. We found that overexpression of α-1,6-fucosyltransferase (Fut8) inhibited hemoglobin production in the MEL cell. We then performed functional analysis of FUT8 to understand the mechanism of suppression of hemoglobin production and erythroid differentiation, as observed in MEL and K562 cells.

      DISCUSSION

      DNA microarray is a powerful tool to understand biological phenomena regulated by multiple genes, such as cell differentiation, because it can comprehensively analyze gene expression at one time point. However, it also detects the noise of gene expression, such as responses to the stresses of chemical addition and cell proliferation that occur during chemical induction of the differentiation of cell lines. In this study, we compensated for the shortcomings of our microarray experiments by comparing the expression profiles of HD and LD MEL cells. The only difference between these cells was their ability to differentiate in response to chemicals that induce cell differentiation. We also effectively selected for differentiation-related genes by focusing on the genes consistently regulated among the three chemical inducers.
      Next, we evaluated the direct relationship between the targeted genes and differentiation by gain-of-function, which was performed using an overexpression study. We observed that Fut8 overexpression inhibited hemoglobin production during MEL cell differentiation; the same effect was observed in human K562 cells.
      Fut8 is a member of the fucosyltransferase gene family. Fucosyltransferases are involved in various biological and pathological processes in eukaryotic organisms, including tissue development, angiogenesis, fertilization, cell adhesion, inflammation, and tumor metastasis (
      • Ma B.
      • Simala-Grant J.L.
      • Taylor D.E.
      Fucosylation in prokaryotes and eukaryotes.
      ). There are two main types of fucosyltransferases in the mouse. One of the main types of fucosyltransferases catalyzes fucosylation of N-linked glycans at asparagine residues. FUT1 and FUT2 transfer fucose to galactose in an α-1,2 linkage. FUT3 and FUT5 transfer fucose to N-acetylglucosamine in an α-1,3 or α-1,4 linkage. FUT4, FUT6, FUT7, FUT9, FUT10, and FUT11 transfer fucose to N-acetylglucosamine in an α-1,3 linkage. Finally, FUT8 transfers fucose to N-acetylglucosamine in an α-1,6 linkage. Another main type of fucosyltransferase in mice catalyzes fucosylation of O-linked glycans at serine or threonine residues. There are two O-fucosyltransferases, POFUT1 and POFUT2. In this study, FUT8 was the only fucosyltransferase that showed inhibition of hemoglobin production. This indicates that core fucosylation plays a key role in hemoglobin production and cell differentiation because FUT8 is the only fucosyltransferase that adds fucose to the core asparagine-linked glycans.
      To evaluate whether FUT8-mediated core fucosylation directly affects hemoglobin production, we performed mutagenesis studies of the donor substrate-binding domain and the flexible loop that are essential for core fucosylation activity. We observed that FUT8-mediated inhibition of hemoglobin production was cancelled by these FUT8 mutations in MEL cells. Moreover, the same results were observed in K562 cells. This is the first report that the regulation of core fucosylation is essential for hemoglobin production and erythroid differentiation. In the development of other tissues, it has previously been reported that FUT8-mediated core fucosylation of signal-transducing receptors positively regulates the function of the receptors. However, there have been no reports of FUT8-mediated negative regulation of differentiation. Fut8 knock-out mice lack core fucosylation in both their epidermal growth factor receptors and platelet-derived growth factor receptors, and they experience early death during postnatal development (∼70% Fut8 null mice) or growth retardation (
      • Wang X.
      • Gu J.
      • Ihara H.
      • Miyoshi E.
      • Honke K.
      • Taniguchi N.
      Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling.
      ). The lack of core fucosylation of transforming growth factor β1 (TGF-β) receptors led to abnormal lung development and an emphysema-like phenotype in Fut8 null mice (
      • Wang X.
      • Inoue S.
      • Gu J.
      • Miyoshi E.
      • Noda K.
      • Li W.
      • Mizuno-Horikawa Y.
      • Nakano M.
      • Asahi M.
      • Takahashi M.
      • Uozumi N.
      • Ihara S.
      • Lee S.H.
      • Ikeda Y.
      • Yamaguchi Y.
      • Aze Y.
      • Tomiyama Y.
      • Fujii J.
      • Suzuki K.
      • Kondo A.
      • Shapiro S.D.
      • Lopez-Otin C.
      • Kuwaki T.
      • Okabe M.
      • Honke K.
      • Taniguchi N.
      Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.
      ). In B cell development, the loss of core fucosylation in both α4β1 integrin and vascular cell adhesion molecule 1 led to a decrease in binding between pre-B cells and stromal cells, which impaired the generation of pre-B cells in Fut8 null mice (
      • Li W.
      • Ishihara K.
      • Yokota T.
      • Nakagawa T.
      • Koyama N.
      • Jin J.
      • Mizuno-Horikawa Y.
      • Wang X.
      • Miyoshi E.
      • Taniguchi N.
      • Kondo A.
      Reduced α4β1 integrin/VCAM-1 interactions lead to impaired pre-B cell repopulation in α1,6-fucosyltransferase deficient mice.
      ). There were no reports about Fut8 null mice of defective of erythroid phenotypes, because it seem that down-regulation of Fut8 promotes erythroid differentiation. These reports predict that signal-transducing receptors other than EpoR may play a role in erythroid differentiation and that core fucosylation of these receptors may play a key part in this role. We think the TGF-β receptor is one of the candidate targets of FUT8. This receptor was identified as the target of core fucosylation (
      • Wang X.
      • Inoue S.
      • Gu J.
      • Miyoshi E.
      • Noda K.
      • Li W.
      • Mizuno-Horikawa Y.
      • Nakano M.
      • Asahi M.
      • Takahashi M.
      • Uozumi N.
      • Ihara S.
      • Lee S.H.
      • Ikeda Y.
      • Yamaguchi Y.
      • Aze Y.
      • Tomiyama Y.
      • Fujii J.
      • Suzuki K.
      • Kondo A.
      • Shapiro S.D.
      • Lopez-Otin C.
      • Kuwaki T.
      • Okabe M.
      • Honke K.
      • Taniguchi N.
      Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.
      ), and TGF-β induces hemoglobin production in K562 and other human erythroleukemia cells (
      • Burger P.E.
      • Dowdle E.B.
      • Lukey P.T.
      • Wilson E.L.
      Basic fibroblast growth factor antagonizes transforming growth factor β-mediated erythroid differentiation in K562 cells.
      ,
      • Kaneko K.
      • Furuyama K.
      • Aburatani H.
      • Shibahara S.
      Hypoxia induces erythroid-specific 5-aminolevulinate synthase expression in human erythroid cells through transforming growth factor-β signaling.
      ).
      FUT8 knockdown studies using shRNA in K562 cells indicated that the decrease in FUT8 expression was important for hemoglobin production and differentiation. In the microarray experiments, we also observed that Fut8 expression was decreased during HD MEL cell differentiation. Our results indicate that Fut8 is regulated downstream of Myc and Myb. Both transcription factors were already known as regulators of erythroid differentiation (
      • Pang C.J.
      • Lemsaddek W.
      • Alhashem Y.N.
      • Bondzi C.
      • Redmond L.C.
      • Ah-Son N.
      • Dumur C.I.
      • Archer K.J.
      • Haar J.L.
      • Lloyd J.A.
      • Trudel M.
      Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis.
      ,
      • Jayapal S.R.
      • Lee K.L.
      • Ji P.
      • Kaldis P.
      • Lim B.
      • Lodish H.F.
      Down-regulation of Myc is essential for terminal erythroid maturation.
      ,
      • Rylski M.
      • Welch J.J.
      • Chen Y.Y.
      • Letting D.L.
      • Diehl J.A.
      • Chodosh L.A.
      • Blobel G.A.
      • Weiss M.J.
      GATA-1-mediated proliferation arrest during erythroid maturation.
      ,
      • Bartnek P.
      • Králová J.
      • Blendinger G.
      • Dvorák M.
      • Zenke M.
      GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter.
      ,
      • Kaneko-Ishino T.
      • Kume T.U.
      • Sasaki H.
      • Obinata. M.
      • Oishi M.
      Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.
      ,
      • Todokoro K.
      • Watson R.J.
      • Higo H.
      • Amanuma H.
      • Kuramochi S.
      • Yanagisawa H.
      • Ikawa Y.
      Down-regulation of c-myb gene expression is a prerequisite for erythropoietin-induced erythroid differentiation.
      ). The upstream sequence of human FUT8 has binding sites for MYB and GATA-1 (
      • Yamaguchi Y.
      • Ikeda Y.
      • Takahashi T.
      • Ihara H.
      • Tanaka T.
      • Sasho C.
      • Uozumi N.
      • Yanagidani S.
      • Inoue S.
      • Fujii J.
      • Taniguchi N.
      Genomic structure and promoter analysis of the human α1,6-fucosyltransferase gene (FUT8).
      ). In a study exploring the regulation of erythroid gene expression by GATA-1, Fut8 expression was rapidly down-regulated after GATA-1 induction (
      • Welch J.J.
      • Watts J.A.
      • Vakoc C.R.
      • Yao Y.
      • Wang H.
      • Hardison R.C.
      • Blobel G.A.
      • Chodosh L.A.
      • Weiss M.J.
      Global regulation of erythroid gene expression by transcription factor GATA-1.
      ). This result also indicates that Fut8 expression is down-regulated downstream of GATA-1.
      In contrast, the enzyme activity of FUT8 was increased during megakaryocytic differentiation (
      • Bany-Łaszewicz U.
      • Kamińska J.
      • Klimczak-Jajor E.
      • Kocielak J.
      The activity of α1,6-fucosyltransferase during human megakaryocytic differentiation.
      ). This fact is the opposite to our findings that a decrease in FUT8 progressed differentiation of pre-erythroblasts to basophilic normoblasts and polychromatic erythroblasts. FUT8 may be involved in the decision whether MEPs differentiate into erythrocytes or megakaryocytes, a role similar to FLI-1, which suppresses erythroid differentiation but activates megakaryocytic differentiation.
      In conclusion, expression profile analysis of the differential differentiation ability of MEL cells using DNA microarrays identified FUT8 as a suppressor of differentiation and demonstrated that FUT8-mediated core fucosylation regulates hemoglobin production and erythroid differentiation. In a previous erythropoiesis study, several transcription factors and cytokines, including GATA-1 and erythropoietin, were shown to regulate hemoglobin production and erythroid differentiation. In addition, our findings clearly demonstrate that modification of proteins by glycosylation is important for hemoglobin production and erythroid differentiation. We are currently exploring the target proteins of FUT8-mediated core fucosylation. Further study of FUT8-mediated core fucosylation during erythroid differentiation will help to clarify the additional details of the mechanism of differentiation.

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