Intestinal DMT1 Cotransporter Is Down-regulated by Hepcidin via Proteasome Internalization and Degradation

• Published in: Gastroenterology (New York, N.Y. 1943) Vol. 140; no. 4; pp. 1261 - 1271.e1
• Main Authors: Brasse–Lagnel, Carole, Karim, Zoubida, Letteron, Philippe, Bekri, Soumeya, Bado, André, Beaumont, Carole
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2011
• Subjects: Animals; Antimicrobial Cationic Peptides - metabolism; Caco-2 Cells; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Down-Regulation - physiology; Duodenum - metabolism; Gastroenterology and Hepatology; Gene Expression - physiology; Green Fluorescent Proteins - genetics; Hemochromatosis; Hepcidin; Hepcidins; Homeostasis - physiology; Humans; Intestinal Mucosa - metabolism; Iron - metabolism; Iron Overload; Iron Radioisotopes - pharmacokinetics; Male; Mice; Mice, Inbred C57BL; Proteasome Endopeptidase Complex - metabolism; Proteolysis; Ubiquitination - physiology; PBS; Hemochromatosis; HIF; FPN; ferroportin; hypoxia-inducible factor; mRNA; phosphate-buffered saline; phenylmethylsulfonyl fluoride; green fluorescent protein; IL-6; Interleukin-6; GFP; Hank's balance salt solution; messenger RNA; Proteolysis; PMSF; protease inhibitors; PI; HBSS; iron-responsive element; Hepcidin; IRE; Iron Overload
• ISSN: 0016-5085; 1528-0012
• DOI: 10.1053/j.gastro.2010.12.037

Backgrounds & Aims 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 using55 FeNTA. 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 of55 Fe 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.