title: Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation.
abstract: OBJECTIVE
  The mechanism by which hepcidin regulates iron export from macrophages has been well established and is believed to involve degradation of ferroportin. However, in the small intestine, hepcidin's mechanisms of action are not known. We studied human polarized intestinal (Caco-2/TC7) cells and mouse duodenal segments, ex vivo, to investigate the molecular mechanisms by which hepcidin down-regulates intestinal transepithelial iron transport.
  
  
  METHODS
  Iron transport was analyzed using ⁵⁵FeNTA. Expression of Divalent Metal Transporter 1 (DMT1) and ferroportin was evaluated by reverse-transcription quantitative polymerase chain reaction and immunoblotting. Videomicroscopy analysis was performed on live cells that expressed either DMT1 or ferroportin fused to green fluorescent protein.
  
  
  RESULTS
  In Caco-2/TC7 cells, physiologic doses of hepcidin (50-1000 nmol/L) inhibited transport of ⁵⁵Fe in a dose-dependent manner; a half-maximum effect was observed at 75-100 nmol/L. However, 200 nmol/L hepcidin induced a significant decrease in DMT1 protein expression but no change in ferroportin protein levels, unlike macrophages. This result was confirmed ex vivo in isolated duodenal segments: 200 nmol/L hepcidin induced a significant reduction in iron transport and DMT1 protein levels but no change in ferroportin levels. In Caco-2/TC7 cells, the effect of hepcidin on the DMT1 protein level was completely abolished in the presence of a proteasome inhibitor (MG-132); DMT1 ubiquitination was induced by the addition of hepcidin.
  
  
  CONCLUSIONS
  An acute increase in hepcidin concentration reduces intestinal iron absorption through ubiquitin-dependent proteasome degradation of DMT1.
authors.affiliation: INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Université Paris Diderot, site Bichat, Paris, France. carole.lagnel@univ-rouen.fr
authors.forename: Carole
authors.initials: C
authors.lastname: Brasse-Lagnel
authors.forename: Zoubida
authors.initials: Z
authors.lastname: Karim
authors.forename: Philippe
authors.initials: P
authors.lastname: Letteron
authors.forename: Soumeya
authors.initials: S
authors.lastname: Bekri
authors.forename: André
authors.initials: A
authors.lastname: Bado
authors.forename: Carole
authors.initials: C
authors.lastname: Beaumont
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism*; D018938:Caco-2 Cells; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology; D004386:Duodenum / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D064451:Hepcidins; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007504:Iron Radioisotopes / Q000493:pharmacokinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology; D000098742:Ferroportin; D000099236:Solute Carrier Family 11, Member 2
chemical_list: D023181:Antimicrobial Cationic Peptides; D027682:Cation Transport Proteins; D049452:Green Fluorescent Proteins; D064451:Hepcidins; D007501:Iron; D007504:Iron Radioisotopes; D046988:Proteasome Endopeptidase Complex; D000098742:Ferroportin; D000099236:Solute Carrier Family 11, Member 2; C578702:HAMP protein, human; C579599:Hamp protein, mouse