Biological Trace Element Research

, Volume 146, Issue 2, pp 281–286 | Cite as

Iron, Copper, and Zinc Transport: Inhibition of Divalent Metal Transporter 1 (DMT1) and Human Copper Transporter 1 (hCTR1) by shRNA

  • Alejandra Espinoza
  • Solange Le Blanc
  • Manuel Olivares
  • Fernando Pizarro
  • Manuel Ruz
  • Miguel Arredondo


Iron (Fe), copper (Cu), and zinc (Zn) fulfill various essential biological functions and are vital for all living organisms. They play important roles in oxygen transport, cell growth and differentiation, neurotransmitter synthesis, myelination, and synaptic transmission. Because of their role in many critical functions, they are commonly used in food fortification and supplementation strategies globally. To determine the involvement of divalent metal transporter 1 (DMT1) and human copper transporter 1 (hCTR1) on Fe, Cu, and Zn uptake, Caco-2 cells were transfected with four different shRNA plasmids to selectively inhibit DMT1 or hCTR1 transporter expression. Fe and Cu uptake and total Zn content measurements were performed in shRNA-DMT1 and shRNA-hCTR1 cells. Both shRNA-DMT1 and shRNA-hCTR1 cells had lower apical Fe uptake (a decrease of 51% and 41%, respectively), Cu uptake (a decrease of 25.8% and 38.5%, respectively), and Zn content (a decrease of 23.1% and 22.7%, respectively) compared to control cells. These results confirm that DMT1 is involved in active transport of Fe, Cu, and Zn although Zn showed a different relative capacity. These results also show that hCTR1 is able to transport Fe and Zn.


Iron Copper Zinc DMT1 hCTR1 shRNA 


  1. 1.
    Crichton R, Boelaert JR, Braun V et al (2001) The importance of iron for biological systems. In: Crichton R (ed) Inorganic biochemistry of iron metabolism: from molecular mechanisms to clinical consequences, 2nd edn. Wiley, ChichesterGoogle Scholar
  2. 2.
    McCall KA, Huang C, Fierke CA (2000) Function and mechanism of zinc of zinc metalloenazymes. J Nutr 130:1437S–1446SPubMedGoogle Scholar
  3. 3.
    Institute of Medicine, Food and Nutrition Board (2002) Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. National Academy Press, Washington, pp 224–257Google Scholar
  4. 4.
    Ruz M (2003) Zinc properties and determination. In: Caballero B, Trugo L, Finglas P (eds) Encyclopedia of food sciences and nutrition. Academic, London, pp 6267–6272CrossRefGoogle Scholar
  5. 5.
    López de Romaña D, Olivares M, Uauy R, Araya M (2011) Risks and benefits of copper in light of new insights of copper homeostasis. J Trace Elem Med Biol 25:3–13CrossRefGoogle Scholar
  6. 6.
    Gunshin H, Mackenzie B, Berger U et al (1997) Cloning and characterization of a mammalian proton-coupled metal-iron transporter. Nature 388:482–488PubMedCrossRefGoogle Scholar
  7. 7.
    Arredondo M, Muñoz P, Mura C et al (2003) DMT1, a physiologically relevant apical Cu+1 transporter of intestinal cells. Am J Physiol 284:C1525–C1530Google Scholar
  8. 8.
    Zhou B, Gitschier A (1997) hCTR1: a human gene for copper uptake identified by complementation in yeast. Proc Natl Acad Sci 94:7481–7486PubMedCrossRefGoogle Scholar
  9. 9.
    Foster M, Samman S (2010) Zinc and redox signaling: perturbations associated with cardiovascular disease and diabetes mellitus. Antioxid Redox Signal 13:1549–1573PubMedCrossRefGoogle Scholar
  10. 10.
    Ramakrishnan U (2002) Prevalence of micronutrient malnutrition worldwide. Nutr Rev 60(5):S46–S52PubMedCrossRefGoogle Scholar
  11. 11.
    Olivares M, Walter T, Hertrampf E (1999) Anaemia and iron deficiency disease in children. Brit Med Bull 55:534–548PubMedCrossRefGoogle Scholar
  12. 12.
    Ruz M, Cavan KR, Bettger WJ et al (1991) Development of a dietary model for the study of mild zinc deficiency in humans and evaluation of some biochemical and functional indices of zinc status. Am J Clin Nutr 53:1295–1203Google Scholar
  13. 13.
    Ruz M, Cavan KR, Bettger WJ et al (1992) Erythrocytes, erythrocyte membranes, neuthrophils, an platelets as biopsy materials for the assessment of zinc status in humans. Br J Nutr 68:515–527PubMedCrossRefGoogle Scholar
  14. 14.
    International Zinc Nutrition Consultative Group (IZiNCG), Brown KH, Rivera JA et al (2004) International Zinc Nutrition Consultative Group (IZiNCG) technical document #1. Assessment of the risk of zinc deficiency in populations and options for its control. Food Nutr Bull 25:S99–S203PubMedGoogle Scholar
  15. 15.
    Benoist B, Darnton-Hil I, Davidsson L, Fontaine O, Hotz C (2007) Conclusions of the Joint WHO/UNICEF/IAEA/IZiNCG Interagency Meeting on Zinc Status Indicators. Food Nutr Bull 28:S480–S484PubMedGoogle Scholar
  16. 16.
    INACG (1977) Guidelines for the eradication of iron deficiency anemia, a report of the International Nutritional Anemia Consultative Group. INACG, Washington, pp 1–29Google Scholar
  17. 17.
    Olivares M, Pizarro F, Ruz M (2007) Zinc inhibits nonheme iron bioavailability in humans. Biol Trace Elem Res 117:7–14PubMedCrossRefGoogle Scholar
  18. 18.
    Yamaji S, Tennant J, Tandy S et al (2001) Zinc regulates the function and expression of the iron transporters DMT1and IREG1 in human intestinal Caco-2 cells. FEBS Lett 507:137–141PubMedCrossRefGoogle Scholar
  19. 19.
    Arredondo M, Martínez R, Núñez MT, Ruz M, Olivares M (2006) Inhibition of iron and copper uptake by iron, copper and zinc. Biol Res 39:95–102PubMedCrossRefGoogle Scholar
  20. 20.
    Olivares M, Pizarro F, López de Romaña D et al (2010) Acute copper supplementation does not inhibit non-heme iron bioavalability in humans. Biol Trace Elem Res 136:180–186PubMedCrossRefGoogle Scholar
  21. 21.
    Tallkvist J, Bowlus CL, Lönnerdal B (2000) Functional and molecular responses of human intestinal Caco-2 cells to iron treatment. Am J Clin Nutr 72:770–775PubMedGoogle Scholar
  22. 22.
    Tandy S, Williams M, Leggett A et al (2000) Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells. J Biol Chem 275:1023–1029PubMedCrossRefGoogle Scholar
  23. 23.
    Kordas K, Stoltzfus RJ (2004) New evidence of iron and zinc interplay at the enterocyte and neural tissues. J Nutr 134:1295–1298PubMedGoogle Scholar
  24. 24.
    Wang K, Zhou B, Kuo YM, Zemansky J, Gitschier J (2002) A novel member of a zinc transporter family is defective in acrodermatitis enteropathica. Am J Hum Genet 71:66–73PubMedCrossRefGoogle Scholar
  25. 25.
    Pfaffl M (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45PubMedCrossRefGoogle Scholar
  26. 26.
    Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  27. 27.
    Zimnicka A, Maryon E, Kaplan J (2007) Human copper transportes hCTR1 mediates basolateral uptake of copper into enterocytes. Implications for copper homeostasis. J Biol Chem 282:26471–26480PubMedCrossRefGoogle Scholar
  28. 28.
    Iyengar V, Pullakhandam R, Nair KM (2009) Iron-zinc interaction during uptake in human intestinal Caco-2 cell line: kinetic analyses and possible mechanism. Indian J Biochem Biophys 46(4):299–306PubMedGoogle Scholar
  29. 29.
    Liuzzi JP, Cousins R (2004) Mammalian zinc transporters. Annu Rev Nutr 24:151–172PubMedCrossRefGoogle Scholar
  30. 30.
    Qin Y, Dittmer PJ, Park JG, Jansen KB, Palmer AE (2011) Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2þ with genetically encoded sensors. PNAS 108:7351–7356PubMedCrossRefGoogle Scholar
  31. 31.
    Garrick MD, Singleton ST, Vargas F et al (2006) DMT1: which metals does it transport? Biol Res 39:79–85PubMedCrossRefGoogle Scholar
  32. 32.
    Liuzzi JP, Aydemir F, Nam H, Knutson M, Cousins R (2006) Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells. Proc Natl Acad Sci U S A 103(37):13612–13617PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Alejandra Espinoza
    • 1
  • Solange Le Blanc
    • 1
  • Manuel Olivares
    • 1
  • Fernando Pizarro
    • 1
  • Manuel Ruz
    • 2
  • Miguel Arredondo
    • 1
  1. 1.Micronutrient Laboratory, Institute of Nutrition and Food TechnologyUniversity of ChileMaculChile
  2. 2.Department of Nutrition, Faculty of MedicineUniversity of ChileSantiagoChile

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