Augmented internalisation of ferroportin to late endosomes impairs iron uptake by enterocyte-like IEC-6 cells.
Absorption of iron occurs by duodenal enterocytes, involving uptake by the divalent metal transporter-1 (DMT1) and release by ferroportin. Ferroportin responds to the hepatocyte-produced 25-amino-acid-peptide hepcidin-25 by undergoing internalisation to late endosomes that impair iron release. Ferroportin is also expressed on the apical membrane of polarised Caco-2 cells, rat intestinal cells and in IEC-6 cells (an intestinal epithelial cell line). A blocking antibody to ferroportin also impairs the uptake, but not the release, of iron. In this study IEC-6 cells were used to study the mechanism of impairment or recovery from impairment produced by the blocking antibody and the fate of DMT1 and ferroportin. Uptake of 1 muM Fe(II) was studied by adding the antibody from time 0 and after adding or removing the antibody once a steady state had been reached. Surface binding, maximum iron transport rate V(max) and transporter affinity (K(m)) were measured after impairment of iron uptake. Ferroportin and DMT1 distribution were assessed by immunofluorescence microscopy. Antibody-mediated impairment, or recovery from impairment, of Fe(II) uptake occurred within minutes. Impairment was lost when the antibody was combined with the immunizing peptide. DMT1 and ferroportin undergo internalisation to late endosomes and, in the presence of the antibody, augmented internalisation of DMT1 and ferroportin caused swelling of late endosomes. Surface binding of Fe(II) and iron transport V(max) were reduced by 50%, indicating that the antibody removed membrane-bound DMT1. The ferroportin antibody induced rapid turnover of membrane ferroportin and DMT1 and its internalisation to late endosomes, resulting in impaired Fe(II) uptake.
Oates
Phillip S
PS
Department of Physiology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. poates@cyllene.uwa.edu.au
Thomas
Carla
C
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D051381:Rats; D000098742:Ferroportin; D000099236:Solute Carrier Family 11, Member 2
D000906:Antibodies; D027682:Cation Transport Proteins; D007501:Iron; D033862:Iron-Binding Proteins; D000098742:Ferroportin; D000099236:Solute Carrier Family 11, Member 2