scholarly journals Comparison of mammalian cell lines expressing distinct isoforms of divalent metal transporter 1 in a tetracycline-regulated fashion

2006 ◽  
Vol 398 (3) ◽  
pp. 539-546 ◽  
Author(s):  
Michael D. Garrick ◽  
Hung-Chieh Kuo ◽  
Farida Vargas ◽  
Steven Singleton ◽  
Lin Zhao ◽  
...  
Keyword(s):  
Cell Lines ◽  
Iron Uptake ◽  
Divalent Metal ◽  
Maximal Response ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Doxycycline Treatment ◽  
Mammalian Cell Lines ◽  
Toxicological Studies

DMT1 (divalent metal transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; ±IRE). We have compared 1A/+IRE and 2/−IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was ∼10-fold greater than that of untreated cells, while expression in the untreated cells was ∼5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at ∼1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at ∼5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.

scholarly journals The Divalent Metal Transporter 1 (DMT1) Is Required for Iron Uptake and Normal Development of Oligodendrocyte Progenitor Cells

2018 ◽  
Vol 38 (43) ◽  
pp. 9142-9159 ◽  
Author(s):  
Veronica T. Cheli ◽  
Diara A. Santiago González ◽  
Leandro N. Marziali ◽  
Norma N. Zamora ◽  
María E. Guitart ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Iron Uptake ◽  
Normal Development ◽  
Divalent Metal ◽  
Oligodendrocyte Progenitor Cells ◽  
Divalent Metal Transporter 1 ◽  
Oligodendrocyte Progenitor ◽  
Divalent Metal Transporter ◽  
Metal Transporter

scholarly journals Small molecule inhibitors of divalent metal transporter-1

2009 ◽  
Vol 296 (4) ◽  
pp. G798-G804 ◽  
Author(s):  
Peter D. Buckett ◽  
Marianne Wessling-Resnick
Keyword(s):  
Small Molecule ◽  
Iron Metabolism ◽  
Iron Uptake ◽  
Small Molecule Inhibitors ◽  
Structural Features ◽  
Divalent Metal ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Copper Chelation ◽  
Metal Transporter

Divalent metal transporter-1 (DMT1) is a divalent cation transporter that plays a key role in iron metabolism by mediating ferrous iron uptake across the small intestine. We have previously identified several small molecule inhibitors of iron uptake ( 4 ). Using a cell line that stably overexpresses DMT1, we screened the ability of these inhibitors to specifically block this transporter's activity. One compound, NSC306711, inhibited DMT1-mediated iron uptake in a reversible and competitive manner. This inhibitor is a polysulfonated dye containing two copper centers. Although one of these two sites could be chelated by Triethylenetetramine copper chelation did not perturb NSC306711 inhibition of DMT1 activity. Several other polysulfonated dyes with structural features similar to NSC306711 were identified as potential DMT1 transport inhibitors. This study characterizes important pharmacological tools that can be used to probe DMT1's mechanism of iron transport and its role in iron metabolism.

scholarly journals Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells

2003 ◽  
Vol 284 (1) ◽  
pp. C44-C50 ◽  
Author(s):  
Desmond I. Bannon ◽  
Roger Abounader ◽  
Peter S. J. Lees ◽  
Joseph P. Bressler
Keyword(s):  
Cell Lines ◽  
Toxic Metals ◽  
Control Cell ◽  
Divalent Metal ◽  
Lead Uptake ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Ph Range ◽  
Cadmium And Lead

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller Vmaxfor iron uptake compared with control cell lines. One clone was further characterized and found to display an ∼50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.

Cytokine-mediated regulation of iron transport in human monocytic cells

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4148-4154 ◽  
Author(s):  
Susanne Ludwiczek ◽  
Elmar Aigner ◽  
Igor Theurl ◽  
Günter Weiss
Keyword(s):  
Iron Uptake ◽  
Iron Transport ◽  
Divalent Metal ◽  
Mrna Levels ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Regulatory Pathways ◽  
Metal Transporter ◽  
Cellular Expression ◽  
Ifn Γ

Abstract Under chronic inflammatory conditions cytokines induce a diversion of iron traffic, leading to hypoferremia and retention of the metal within the reticuloendothelial system. However, the regulatory pathways underlying these disturbances of iron homeostasis are poorly understood. We investigated transferrin receptor (TfR)–dependent and –independent iron transport mechanisms in cytokine-stimulated human monocytic cell lines THP-1 and U937. Combined treatment of cells with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) reduced TfR mRNA levels, surface expression, and iron uptake, and these effects were reversed by interleukin-10 (IL-10), thus stimulating TfR-mediated iron acquisition. IFN-γ and LPS dose-dependently increased the cellular expression of divalent metal transporter-1, a transmembrane transporter of ferrous iron, and stimulated the uptake of nontransferrin bound iron (NTBI) into cells. At the same time, IFN-γ and LPS down-regulated the expression of ferroportin mRNA, a putative iron exporter, and decreased iron release from monocytes. Preincubation with IL-10 partly counteracted these effects. Our results demonstrate that the proinflammatory stimuli IFN-γ and LPS increase the uptake of NTBI via stimulation of divalent metal transporter-1 expression and cause retention of the metal within monocytes by down-regulating ferroportin synthesis. Opposite, the anti-inflammatory cytokine IL-10 stimulates TfR-mediated iron uptake into activated monocytes. The regulation of iron transport by cytokines is a key mechanism in the pathogenesis of anemia of chronic disease and a promising target for therapeutic intervention.

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