Considering that the development of hepatic lesions related to iron overload diseases might be a result of abnormally expressed hepatic genes, we searched for new genes up-regulated under the condition of iron excess. By suppressive subtractive hybridization performed between livers from carbonyl iron-overloaded and control mice, we isolated a 225-base pair cDNA. By Northern blot analysis, the corresponding mRNA was confirmed to be overexpressed in livers of experimentally (carbonyl iron and iron-dextran-treated mice) and spontaneously (␤ 2 -microglobulin knockout mice) ironoverloaded mice. In addition, ␤ 2 -microglobulin knockout mice fed with a low iron content diet exhibited a decrease of hepatic mRNA expression. The murine fulllength cDNA was isolated and was found to encode an 83-amino acid protein presenting a strong homology in its C-terminal region to the human antimicrobial peptide hepcidin. In addition, we cloned the corresponding rat and human orthologue cDNAs. Both mouse and human genes named HEPC are constituted of 3 exons and 2 introns and are located on chromosome 7 and 19, respectively, in close proximity to USF2 gene. In mouse and human, HEPC mRNA was predominantly expressed in the liver. During both in vivo and in vitro studies, HEPC mRNA expression was enhanced in mouse hepatocytes under the effect of lipopolysaccharide. Finally, to analyze the intracellular localization of the predicted protein, we used the green fluorescent protein chimera expression vectors. The murine green fluorescent protein-prohepcidin protein was exclusively localized in the nucleus. When the putative nuclear localization signal was deleted, the resulting protein was addressed to the cytoplasm. Taken together, our data strongly suggest that the product of the new liver-specific gene HEPC might play a specific role during iron overload and exhibit additional functions distinct from its antimicrobial activity.

Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts were very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoterluciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein ␣ (C/EBP␣) and C/EBP␤ were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4␣ (HNF4␣) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (؊250/؊230) predominantly bound C/EBP␣ from rat liver nuclear extracts. Hepatic deletion of the C/EBP␣ gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4␣-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBP␣ was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBP␣ protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBP␣ is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.

Hepcidin, a key regulator of iron metabolism, is synthesized by the liver. Hepcidin binds to the iron exporter ferroportin to regulate the release of iron into plasma from macrophages, hepatocytes, and enterocytes. We analyzed liver samples from patients undergoing hepatic surgery for cancer or receiving liver transplants and IntroductionHepcidin is a peptide found in human plasma and urine 1,2 and synthesized in the liver. 1-3 Studies in mice, humans, and cell cultures demonstrated that hepcidin mRNA levels were regulated by iron stores, 3 inflammation, 3,4-6 anemia, and hypoxia, 4 and influenced by the mouse strain and sex and transcription factors involved in hepatocyte differentiation. 7,8 Hepcidin-deficient mice accumulated iron in the liver and pancreas 9 and mice engineered to overexpress hepcidin were born with severe iron deficiency that was not corrected by iron supplementation. 10 Taken together these data suggested that hepcidin was a hormone involved in the regulation of iron metabolism. 9 Studies of patients with iron disorders demonstrated the involvement of hepcidin in regulation of iron metabolism during juvenile hemochromatosis, 11 classical HFE-related hemochromatosis, 12,13 and chronic inflammatory diseases. 5,14 Hepcidin appears to act by inhibiting cellular iron export through binding directly to the iron exporter ferroportin and inducing its internalization and degradation. 15 Recent understanding of iron metabolism in mice is based on measurements of hepatic hepcidin mRNA, whereas the corresponding human studies examine urinary hepcidin concentrations. We examined the correlation between hepcidin mRNA and urinary hepcidin concentrations in the context of human liver disease. Study design PatientsThirty-six patients, who were operated on for primary or secondary liver carcinoma or received transplants for cirrhosis, were included in this study.The patients with known HFE genetic hemochromatosis, expected to exhibit abnormal hepcidin regulation, 12,16 were excluded. The study was approved by the local ethics committee (CCPPRB) and informed consent was obtained from the patients.From each patient we collected clinical data and serum, urine, and nontumoral liver samples. Liver tissues were frozen in liquid nitrogen or fixed in formalin for histologic studies. The hepatic fibrosis status was evaluated according to the Metavir score 17 ; 20 samples had no or slight fibrosis, 7 samples were moderately fibrotic, and 9 samples were strongly fibrotic or cirrhotic. MethodsClinical laboratory studies. Clinical laboratory assays were performed at the Rennes University Hospital. The liver iron concentration (LIC) was evaluated as described previously. 18 Quantitative RT-PCR. Total RNAs were extracted using the SV Total RNA Isolation System (Promega, Madison, WI). Quality-checked RNA (1 g) was used for reverse transcription (RT) following the manufacturer's protocol (Advantage RT-for-PCR Kit, Clontech, Palo Alto, CA). We performed polymerase chain reactions (PCRs) in triplicate to evaluate the hepcidin...