The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells

C.E. Herbison, K. Thorstensen, Anita Chua, Ross Graham, Peter Leedman, John Olynyk, Debbie Trinder

    Research output: Contribution to journalArticle

    42 Citations (Scopus)

    Abstract

    Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using 59Fe-125I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe2Tf, Co2Tf, and Mn2Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co2Tf and Mn2Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1α protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 μM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15–20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.
    Original languageEnglish
    Pages (from-to)C1567-C1575
    JournalAmerican journal of physiology : cell physiology
    Volume297
    Issue number6
    DOIs
    Publication statusPublished - 2009

    Fingerprint

    Transferrin Receptors
    Transferrin
    Hepatocellular Carcinoma
    Iron
    Coordination Complexes
    Ferritins
    Transfection
    Proteins
    Plasmids
    Up-Regulation
    Transferrins
    Hypoxia-Inducible Factor 1
    Small Interfering RNA
    Real-Time Polymerase Chain Reaction
    Western Blotting
    Metals
    Messenger RNA

    Cite this

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    title = "The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells",
    abstract = "Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using 59Fe-125I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe2Tf, Co2Tf, and Mn2Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co2Tf and Mn2Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1α protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80{\%} while TFR2 knockdown did not affect uptake. At 5 μM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15–20{\%} increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.",
    author = "C.E. Herbison and K. Thorstensen and Anita Chua and Ross Graham and Peter Leedman and John Olynyk and Debbie Trinder",
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    language = "English",
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    The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells. / Herbison, C.E.; Thorstensen, K.; Chua, Anita; Graham, Ross; Leedman, Peter; Olynyk, John; Trinder, Debbie.

    In: American journal of physiology : cell physiology, Vol. 297, No. 6, 2009, p. C1567-C1575.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells

    AU - Herbison, C.E.

    AU - Thorstensen, K.

    AU - Chua, Anita

    AU - Graham, Ross

    AU - Leedman, Peter

    AU - Olynyk, John

    AU - Trinder, Debbie

    PY - 2009

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    N2 - Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using 59Fe-125I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe2Tf, Co2Tf, and Mn2Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co2Tf and Mn2Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1α protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 μM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15–20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.

    AB - Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using 59Fe-125I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe2Tf, Co2Tf, and Mn2Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co2Tf and Mn2Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1α protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 μM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15–20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.

    U2 - 10.1152/ajpcell.00649.2008

    DO - 10.1152/ajpcell.00649.2008

    M3 - Article

    VL - 297

    SP - C1567-C1575

    JO - American Journal of Physiology-Cell Physiology

    JF - American Journal of Physiology-Cell Physiology

    SN - 0363-6143

    IS - 6

    ER -