Use of Nramp2-transfected Chinese hamster ovary cells and reticulocytes from mk/mk mice to study iron transport mechanisms

An-Sheng Zhang, Francois Canonne-Hergaux, Samantha Gruenheid, Philippe Gros, Prem Ponka

Research output: Contribution to journalArticle

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Abstract

Objective: We investigated mechanisms involved in iron (Fe) transport by DMT1 (endosomal Fe(II) exporter, encoded by the Nramp2 gene) using wild-type Chinese hamster ovary (CHO) cells and Nramp2-transfected CHO cells, as well as reticulocytes from normal and mk/mk mice that have a defect in DMT1. Materials and Methods: CHO cells and reticulocytes were incubated with 59Fe bound to various ligands. The radioiron was present in its Fe(II) or Fe(III) forms or bound to transferrin (Tf), and the internalized 59Fe measured under varying experimental conditions. Additionally, 125I-Tf interaction with reticulocytes was investigated and 59Fe incorporation into their heme was determined. Results: Hyperexpression of DMT1 in CHO cells greatly increases their capacity to acquire ferrous iron. Although CHO-Nramp2 cells showed an increase in Fe(III) uptake as compared to CHO cells, they transported Fe(III) with much lower efficacy than Fe(II). In addition to their defect in Fe uptake, mk/mk reticulocytes also showed a decrease in Tf receptor levels. Conclusions: Given that CHO cells acquire iron from Fe(II)-ascorbate with much higher rates than from Fe(III)-Tf, Tf-receptor levels represent the rate-limiting step in their iron uptake. As Fe(III) transport by CHO-Nramp2 cells can be inhibited by the impermeable oxidant K3Fe(CN)6, a membrane ferric reductase is probably needed for reduction of Fe(III) to Fe(II), which is then transported by DMT1. DMT1 is not a limiting factor in Fe acquisition by normal reticulocytes and their heme synthesis.

Original languageEnglish (US)
Pages (from-to)1227-1235
Number of pages9
JournalExperimental Hematology
Volume36
Issue number10
DOIs
StatePublished - Oct 2008

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Reticulocytes
Cricetulus
Ovary
Iron
Transferrin
Transferrin Receptors
Heme
Oxidants
Ligands
Membranes
Genes

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

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Use of Nramp2-transfected Chinese hamster ovary cells and reticulocytes from mk/mk mice to study iron transport mechanisms. / Zhang, An-Sheng; Canonne-Hergaux, Francois; Gruenheid, Samantha; Gros, Philippe; Ponka, Prem.

In: Experimental Hematology, Vol. 36, No. 10, 10.2008, p. 1227-1235.

Research output: Contribution to journalArticle

Zhang, An-Sheng ; Canonne-Hergaux, Francois ; Gruenheid, Samantha ; Gros, Philippe ; Ponka, Prem. / Use of Nramp2-transfected Chinese hamster ovary cells and reticulocytes from mk/mk mice to study iron transport mechanisms. In: Experimental Hematology. 2008 ; Vol. 36, No. 10. pp. 1227-1235.
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abstract = "Objective: We investigated mechanisms involved in iron (Fe) transport by DMT1 (endosomal Fe(II) exporter, encoded by the Nramp2 gene) using wild-type Chinese hamster ovary (CHO) cells and Nramp2-transfected CHO cells, as well as reticulocytes from normal and mk/mk mice that have a defect in DMT1. Materials and Methods: CHO cells and reticulocytes were incubated with 59Fe bound to various ligands. The radioiron was present in its Fe(II) or Fe(III) forms or bound to transferrin (Tf), and the internalized 59Fe measured under varying experimental conditions. Additionally, 125I-Tf interaction with reticulocytes was investigated and 59Fe incorporation into their heme was determined. Results: Hyperexpression of DMT1 in CHO cells greatly increases their capacity to acquire ferrous iron. Although CHO-Nramp2 cells showed an increase in Fe(III) uptake as compared to CHO cells, they transported Fe(III) with much lower efficacy than Fe(II). In addition to their defect in Fe uptake, mk/mk reticulocytes also showed a decrease in Tf receptor levels. Conclusions: Given that CHO cells acquire iron from Fe(II)-ascorbate with much higher rates than from Fe(III)-Tf, Tf-receptor levels represent the rate-limiting step in their iron uptake. As Fe(III) transport by CHO-Nramp2 cells can be inhibited by the impermeable oxidant K3Fe(CN)6, a membrane ferric reductase is probably needed for reduction of Fe(III) to Fe(II), which is then transported by DMT1. DMT1 is not a limiting factor in Fe acquisition by normal reticulocytes and their heme synthesis.",
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AU - Ponka, Prem

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AB - Objective: We investigated mechanisms involved in iron (Fe) transport by DMT1 (endosomal Fe(II) exporter, encoded by the Nramp2 gene) using wild-type Chinese hamster ovary (CHO) cells and Nramp2-transfected CHO cells, as well as reticulocytes from normal and mk/mk mice that have a defect in DMT1. Materials and Methods: CHO cells and reticulocytes were incubated with 59Fe bound to various ligands. The radioiron was present in its Fe(II) or Fe(III) forms or bound to transferrin (Tf), and the internalized 59Fe measured under varying experimental conditions. Additionally, 125I-Tf interaction with reticulocytes was investigated and 59Fe incorporation into their heme was determined. Results: Hyperexpression of DMT1 in CHO cells greatly increases their capacity to acquire ferrous iron. Although CHO-Nramp2 cells showed an increase in Fe(III) uptake as compared to CHO cells, they transported Fe(III) with much lower efficacy than Fe(II). In addition to their defect in Fe uptake, mk/mk reticulocytes also showed a decrease in Tf receptor levels. Conclusions: Given that CHO cells acquire iron from Fe(II)-ascorbate with much higher rates than from Fe(III)-Tf, Tf-receptor levels represent the rate-limiting step in their iron uptake. As Fe(III) transport by CHO-Nramp2 cells can be inhibited by the impermeable oxidant K3Fe(CN)6, a membrane ferric reductase is probably needed for reduction of Fe(III) to Fe(II), which is then transported by DMT1. DMT1 is not a limiting factor in Fe acquisition by normal reticulocytes and their heme synthesis.

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