Iron-induced reactive oxygen species mediate transporter DMT1 endocytosis and iron uptake in intestinal epithelial cells.
Recent evidence shows that iron induces the endocytosis of the iron transporter dimetal transporter 1 (DMT1) during intestinal absorption. We, and others, have proposed that iron-induced DMT1 internalization underlies the mucosal block phenomena, a regulatory response that downregulates intestinal iron uptake after a large oral dose of iron. In this work, we investigated the participation of reactive oxygen species (ROS) in the establishment of this response. By means of selective surface protein biotinylation of polarized Caco-2 cells, we determined the kinetics of DMT1 internalization from the apical membrane after an iron challenge. The initial decrease in DMT1 levels in the apical membrane induced by iron was followed at later times by increased levels of DMT1. Addition of Fe(2+), but not of Cd(2+), Zn(2+), Cu(2+), or Cu(1+), induced the production of intracellular ROS, as detected by 2',7'-dichlorofluorescein (DCF) fluorescence. Preincubation with the antioxidant N-acetyl-l-cysteine (NAC) resulted in increased DMT1 at the apical membrane before and after addition of iron. Similarly, preincubation with the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) resulted in the enhanced presence of DMT1 at the apical membrane. The decrease of DMT1 levels at the apical membrane induced by iron was associated with decreased iron uptake rates. A kinetic mathematical model based on operational rate constants of DMT1 endocytosis and exocytosis is proposed. The model qualitatively captures the experimental observations and accurately describes the effect of iron, NAC, and DMSO on the apical distribution of DMT1. Taken together, our data suggest that iron uptake induces the production of ROS, which modify DMT1 endocytic cycling, thus changing the iron transport activity at the apical membrane.
Iron and Biology of Aging Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile;
Andrés
A
Esparza
Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile; and.
Ziomara P
ZP
Gerdtzen
Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile; and.
Alvaro
A
Olivera-Nappa
Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile; and.
J Cristian
JC
Salgado
Iron and Biology of Aging Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile; mnunez@uchile.cl.
Marco T
http://orcid.org/0000-0002-1967-8570
MT
Núñez
D001692:Biological Transport; D018938:Caco-2 Cells; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology*; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C110547:DMRT1 protein; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D007501:Iron
DMT1; ROS; iron; mathematical modeling; mucosal block