Hepcidin, a recently discovered iron regulatory peptide, is believed to inhibit the release of iron from absorptive enterocytes and macrophages. Liver hepcidin synthesis is induced in vivo by iron stores and inflammation. The molecular basis of the regulation of hepcidin gene expression by these effectors in hepatocytes is currently unknown, although there is strong evidence that indirect mechanisms are involved. The aims of this study were to gain insight into these mechanisms and to determine to what extent other liver cell types are responsible for transducing the signal by which hepcidin expression is regulated in mouse hepatocytes. For this, we depleted Kupffer cells by injection of liposome-encapsulated clodronate and then studied iron-and inflammation-induced hepcidin gene expression. In addition, we directly evaluated the role of the inflammatory cytokine interleukin 6 (IL-6) by using IL-6 -deficient mice. Our results show that iron is able to induce hepcidin gene expression independently of Kupffer cells in the liver and circulating IL-6. In contrast, we show that hepcidin gene induction by inflammation is also independent of Kupffer cells, but involves, at least partly, IL-6. In conclusion, these results show that two independent regulatory pathways control hepcidin gene expression and suggest that hepatocytes play a key role in the regulation of hepcidin gene expression by sensing iron and inflammatory signals. H epcidin is a disulfide-bonded peptide that was first identified as an antimicrobial peptide and was subsequently shown to be a central player in systemic iron homeostasis. Hepcidin is believed to be a negative regulator of dietary iron absorption and iron release by macrophages (for a review, see Ganz 1 ). Complete hepcidin deficiency in mice leads to progressive iron accumulation. This is reminiscent of the phenotype of human hemochromatosis, with predominant iron overload in hepatocytes and iron sparing of the reticuloendothelial cells. 2 Conversely, transgenic animals constitutively expressing the hepcidin gene display iron deficiency anemia. 3 Several mutations in the HAMP gene (human hepcidin) have recently been identified in families with severe juvenile hemochromatosis, confirming the key role of hepcidin in regulating iron homeostasis in humans. [4][5][6][7] The hepcidin gene is expressed predominantly in the liver, where its expression is strictly restricted to hepatocytes. [8][9][10][11] It is synthesized as an 84 -amino acid precursor that is subsequently processed and secreted as a 25-amino acid peptide. This circulating form has been purified from human blood ultrafiltrate and from urine. 9,10 In close correlation with its properties/functions, two main regulators have been shown to control hepcidin gene expression in vivo. First, iron overload, induced experimentally either by iron dextran injection or by iron-rich diet, was found to be associated with an increase in liver hepcidin Abbreviations: IL, interleukin; mRNA, messenger RNA; LPS, lipopolysaccharide; PBS, phosphate-buffe...