Juvenile hemochromatosis is an iron overload disorder caused by mutations in the genes encoding the major iron regulatory hormone hepcidin (HAMP) 1 and hemojuvelin (HFE2)2. We have previously shown that hemojuvelin is a bone morphogenetic protein (BMP) co-receptor and that BMP signals regulate hepcidin expression and iron metabolism3 , 4. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here, we show that in vitro, compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc more potently inhibits hepcidin induction by BMP-2 or BMP-4, but less potently inhibits BMP-6. In vivo, HJV.Fc or a neutralizing BMP-6 antibody inhibits hepcidin expression and increases serum iron, while DRAGON.Fc has no effect. Notably, Bmp6 null mice have a phenotype resembling hereditary hemochromatosis with reduced hepcidin expression and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP-6, and we show that BMP-6 increases hepcidin expression and reduces serum iron in mice. These data support a key role for BMP-6 as a ligand for HJV and an endogenous regulator of hepcidin expression and iron metabolism in vivo.Secreted by the liver 5 , hepcidin inhibits intestinal iron absorption and macrophage iron release by decreasing cell surface expression of the iron exporter ferroportin 6 . Hepcidin is upregulated by iron administration 5,[7][8] and inhibited by anemia 7 . Hepcidin deficiency and unchecked ferroportin activity are the common pathogenic mechanisms underlying the genetic iron Figure 1a, DRAGON.Fc significantly inhibited hepcidin promoter induction by BMP-2 or BMP-4, but was less effective in inhibiting BMP-5, BMP-6, or BMP-7, and did not inhibit BMP-9. In comparison with HJV.Fc, DRAGON.Fc was significantly more potent against BMP-2 (Fig. 1b) and BMP-4 (Fig. 1c), but was less potent against BMP-6 (Fig. 1d). DRAGON.Fc also inhibited endogenous hepcidin mRNA expression in hepatoma-derived HepG2 cells, where basal hepcidin expression is dependent in part on endogenous BMP-2, BMP-4, and BMP-6 ligands 4 ( Supplementary Fig. 1).We then tested whether DRAGON.Fc administration affected hepcidin expression and iron metabolism in vivo. HJV.Fc at a similar dose was used as a positive control. In contrast to HJV.Fc, DRAGON.Fc had no effect on hepatic hepcidin mRNA (Fig. 1e), splenic ferroportin ( Supplementary Fig. 2a-b.), serum iron ( Fig. 1f), serum transferrin saturation, liver iron content, or spleen iron content (Supplementary Fig. 2c-e) compared with mock treated control mice. Anti-BMP-2 activity in the serum of DRAGON.Fc treated mice was confirmed by the ability of this serum to inhibit BMP-2 induction of hepcidin promoter activity in vitro compared with serum from mock treated mice ( Supplementary Fig. 2f).Since DRAGON.Fc had no effect in vivo despite its higher potency in vitro as an inhibitor of BMP-2 and BMP-4 compared with HJV.Fc, and since DRAGON.Fc was less potent at inhibiting BMP-6 compared with HJV.Fc, we hypothesized that the BMP-6 inhibiting p...

Patients with alcoholic liver disease frequently exhibit iron overload in association with increased hepatic fibrosis. Even moderate alcohol consumption elevates body iron stores; however, the underlying molecular mechanisms are unknown. Hepcidin, a circulatory peptide synthesized in the liver, is a key mediator of iron metabolism. Ethanol metabolism significantly down-regulated both in vitro and in vivo hepcidin mRNA and protein expression. 4-Methylpyrazole, a specific inhibitor of the alcohol-metabolizing enzymes, abolished the effects of ethanol on hepcidin. However, ethanol did not alter the expression of transferrin receptor1 and ferritin or the activation of iron regulatory RNA-binding proteins, IRP1 and IRP2. Mice maintained on 10 -20% ethanol for 7 days displayed down-regulation of liver hepcidin expression without changes in liver triglycerides or histology. This was accompanied by elevated duodenal divalent metal transporter1 and ferroportin protein expression. Injection of hepcidin peptide negated the effect of ethanol on duodenal iron transporters. Ethanol down-regulated hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein ␣ (C/EBP␣) but not ␤. Interestingly, the antioxidants vitamin E and N-acetylcysteine abolished both the alcohol-mediated down-regulation of C/EBP␣ binding activity and hepcidin expression in the liver and the up-regulation of duodenal divalent metal transporter 1. Collectively, these findings indicate that alcohol metabolism-mediated oxidative stress regulates hepcidin transcription via C/EBP␣, which in turn leads to increased duodenal iron transport.

Background and Aims-Mutations in HFE are the most common cause of the iron-overload disorder hereditary hemochromatosis (HH). Levels of the main iron regulatory hormone, hepcidin, are inappropriately low in HH mouse models and patients with HFE mutations, indicating that HFE regulates hepcidin. The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is an important endogenous regulator of hepcidin expression. We investigated whether HFE is involved in BMP6-SMAD regulation of hepcidin expression.