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Biological Research
Print version ISSN 0716-9760
Biol. Res. vol.39 no.1 Santiago 2006
http://dx.doi.org/10.4067/S0716-97602006000100022
Biol Res 39: 191-193, 2006 POSTER PRESENTATION / SHORT COMMUNICATION
Regulation of transepithelial transport of iron by hepcidin
NATALIA P MENA, ANDRÉS L ESPARZA and MARCO T NÚÑEZ
Biology Department and Cell Dynamics and Biotechnology Research Center, Faculty of Sciences, Universidad de Chile Dirección para Correspondencia
ABSTRACT
Hepcidin (Hepc) is a 25 amino acid cationic peptide with broad antibacterial and antifungal actions. A likely role for Hepc in iron metabolism was suggested by the observation that mice having disruption of the gene encoding the transcription factor USF2 failed to produce Hepc mRNA and developed spontaneous visceral iron overload. Lately, Hepc has been considered the "stores regulator," a putative factor that signals the iron content of the body to intestinal cells. In this work, we characterized the effect of Hepc produced by hepatoma cells on iron absorption by intestinal cells. To that end, human Hepc cDNA was cloned and overexpressed in HepG2 cells and conditioned media from Hepc-overexpressing cells was used to study the effects of Hepc on intestinal Caco-2 cells grown in bicameral inserts. The results indicate that Hepc released by HepG2 inhibited apical iron uptake by Caco-2 cells, probably by inhibiting the expression of the apical transporter DMT1. These results support a model in which Hepc released by the liver negatively regulates the expression of transporter DMT1 in the enterocyte.
Key terms: hepcidin, iron absorption, DMT1, Caco-2 cells INTRODUCTION
Hepcidin (Hepc) is a 25 amino acid cationic peptide with broad antibacterial and antifungal actions. It is predominantly expressed in the liver and has been detected in blood and urine (Ganz et al., 2004). The human Hepc gene contains three exons that encode a 94-aminoacid pro-hepcidin with a characteristic furin cleavage site immediately N-terminal to the 25-amino acid mature peptide. In addition to the 25-amino acid form, the urine also contains minor 20-and 22-amino-acid forms truncated at the N-terminus (Kulaksiz et al., 2004). Analysis of the sequence of Hepc revealed that the protein contains eight cysteines. Mass spectroscopy and chemical analysis revealed that all of the cysteines are bridged in the sequence, making Hepc a highly constrained peptide (Park et al., 2001). A likely role for Hepc in iron metabolism was suggested by the observation that mice having disruption of the gene encoding the transcription factor USF2 failed to produce Hepc mRNA and developed spontaneous visceral iron overload (Nicolas et al., 2001).Lately, Hepc has been considered the "stores regulator," a putative factor that signals the iron content of the body to intestinal cells. Hepc expression and secretion by the liver correlate directly with hepatic iron levels. Once secreted, Hepc produces inhibition of intestinal iron absorption by the enterocyte. Thus, Hepc mediates a regulatory cycle of iron absorption between the liver and the intestine (reviewed in Andrews, 2004). In this work, we characterized the effect of Hepc produced by hepatoma cells on iron absorption by intestinal cells. To thatend, human Hepc cDNA was cloned and overexpressed in HepG2 cells, and conditioned media from Hepc-overexpressing cells was used to study the effects of Hepc on intestinal Caco-2 cells grown in bicameral inserts.
RESULTS AND DISCUSSION
Production of Hepc by HepG2 cells. A full-length Hepc cDNA was cloned in the pcDNA3 plasmid, and the plasmid was transfected in HepG2 cells. The transfected cells showed an increase of Hepc immunofluorescence. Similarly, Western blot of cell extracts from control and Hepc-transfected cells indicated an increase in a 20 KDa band in transfected cells. 55Fe uptake by Caco-2 cells pre-treated with Hepc-conditioned media. We studied the effects of Hepc on 55Fe absorption by intestinal Caco-2 cells grown in bicameral inserts. Caco-2 cells were incubated for 24 h with control media or media obtained from 72 h-cultures of Hepc-overproducing HepG2 cells. 55Fe uptake from the apical media was performed (Fig. 1). We found that Hepc decreased apical iron uptake, whereas the transfer step to the basolateral media was not affected. The inhibitory effect on apical 55Fe uptake for Hepc was obliterated when the media was first treated with Sepharose-bound anti-Hepc antibody.
Determination of Hepc effect on DMT1 and Ireg1 expression in Caco-2 cells. RNA from Caco-2 cells treated for 48 hours with Hepc-conditioned medium was analyzed for DMT1 and Ireg1 mRNA expression by semi-quantitative RT-PCR. A two- to three-fold decrease in DMT1 mRNA expression was observed in Hepc-treated cells when compared to cells treated with control media. In contrast, no changes in Ireg1 mRNA expression were readily apparent (Mena et al., manuscript in preparation). These results agree with the 55Fe transport experiments, where only apical iron uptake was affected. In summary, the results indicate that Hepc released by HepG2 inhibited apical iron uptake by Caco-2 cells, probably by inhibiting the expression of the apical transporter DMT1. These results support a model in which Hepc released by the liver negatively regulates the expression of transporter DMT1 in the enterocyte. ACKNOWLEDGEMENTS
This work was financed by FONDECYT grant 1040448 and ICM grant P99-031.
REFERENCES
ANDREWS NC (2004) Iron homeostasis and inherited iron overload disorder: An overview. Clin Invest 113: 1251-1253 GANZ T (2004) Hepcidin in iron metabolism. Curr Opin Hematol 11: 251-254 KULAKSIZ H, GEHRKE SG, JANETZKO A, ROST D, BRUCKNER T, KALLINOWSKI B, STREMMEL W (2004) Pro-hepcidin: Expression and cell specific localisation in the liver and its regulation in hereditary haemochromatosis, chronic renal insufficiency, and renal anaemia. Gut 53: 735-743 NICOLAS G, BENNOUN M, DEVAUX I, BEAUMONT C, GRANDCHAMP B, KAHN A, VAULONT S (2001) Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 98: 8780-8785 PARK CH, VALORE EV, WARING AJ, GANZ T (2001) Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 276: 7806-7810 Corresponding author: Marco T. Núñez, Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Santiago, Chile, Tel.: (56-2) 978-7360, Fax: (56-2) 271-2983, E-mail: mnunez@uchile.cl
Received : March 31, 2005. Accepted : May 24, 2005. |