Hepcidin was independently discovered in the years 2000–2001 by various groups. Krause et al. (2000) isolated from human blood ultrafiltrate a 25-residue peptide with antimicrobial activity that they named LEAP-1 (liver-expressed antimicrobial peptide 1). In the same period Pigeon et al. (2001) identified an iron-regulated gene that encoded for the LEAP-1 with high expression in the liver, and much lower expression in the kidney, adipose tissue, heart, and brain. Park et al. (2001) characterized a cysteine-rich peptide and named it hepcidin (hepatic bactericidal protein) for its hepatic origin with a structure typical for an antimicrobial activity. They found homologous cDNAs in the liver of various species from fish to human. A central role of hepcidin in systemic iron homeostasis was soon unambiguously recognized by the finding that inactivation of its gene was associated with severe iron overload in the liver and pancreas (Nicolas et al., 2001). The finding was serendipitous, since the knockout construct was aimed at deleting USF2 gene (upstream stimulatory factor 2) but it also removed the hepcidin adjacent genes. A following specific USF2 knockout mouse had normal hepcidin and iron, while the specific inactivation of hepcidin gene caused iron overload (Nicolas et al., 2002). The importance of hepcidin was conclusively demonstrated by the finding that transgenic mice overexpressing the peptide showed a severe and often lethal anemia (Nicolas et al., 2003). The initial excitement about hepcidin as a central player in the communication of body iron stores to the intestinal absorptive cells (Fleming and Sly, 2001) was further sustained by the finding that patients with homozygous mutations in the hepcidin gene were affected by severe juvenile hemochromatosis (Roetto et al., 2003). The following years were dedicated to the characterization of the gene, its product and the regulation of its expression.

In human the hepcidin gene is in chromosome 19 and encodes a precursor prepropeptide of 84 amino acids that is processed by two sequential cleavages. The first of the signal sequence and the second of the pro-region to produce the mature peptide of 25 amino acids (aa). Furin, a major member of the family of prohormone convertases, is the enzyme involved in the processing, and it recognizes the consensus sequence (QRRRRR↓DTHF) conserved in mammal and fish hepcidins (Shike et al., 2004; Valore and Ganz, 2008; Figure 1A). Chemical or siRNA-mediated inhibition of furin prevents hepcidin maturation but not its secretion from the cell (Valore and Ganz, 2008). The processing may be more complex, since two additional hepcidin N-terminal truncated forms of 22 and 20 aa were originally described (Figure 1A). Hepc-25 and hepc-20 are formed intracellularly and possibly processed in the Golgi apparatus by furin-like proteases and both secreted in the blood. Whereas hepc-22 is present only in urine (Park et al., 2001). The role of hepc-20 and hepc-22 is not clear. The NMR structure of the mature hepcidin 25 was obtained from the refolded synthetic peptide (Jordan et al., 2009). It consists of two short β-strands stabilized by interstrand disulfide bonds (Figure 1B). It has amphipathic properties with cationic charges and hydrophobic surface like most of antimicrobial peptides (Figure 1C), however, the antimicrobial activity of human hepcidin is low and probably not critical. Mouse has two different hepcidin genes (hepc-1 and hepc-2) but only hepc-1 is related with iron metabolism.

Although mammalian hepcidin attracted most interest, it should be mentioned that hepcidin has been described also in fishes, where the size and cysteines are conserved, but the overall amino acid sequence identity with the human one is only 50% (Nemeth and Ganz, 2006). The role of fish hepcidin in systemic iron regulation is unclear.

Most of the regulation of hepcidin expression in the liver occurs at transcriptional level and it is modulated by iron status, inflammation, and hypoxia (Nemeth and Ganz, 2006). Iron supplementation to the animal readily stimulates liver hepcidin mRNA, but this does not occur in cultured cells, of hepatic or non-hepatic lineage, suggesting an indirect mechanism of iron sensing. A breakthrough for the understanding of the regulatory mechanism was the finding that mice with liver conditional inactivation of the SMAD4 gene developed early and severe liver iron overload, since they did not express detectable hepcidin mRNA even after induction with iron and inflammatory stimuli (Wang et al., 2005). SMAD4 is the key player of the TGF-beta/BMP signaling transduction that involves serine kinases, and phosphorylation of SMAD members to form complexes with SMAD4. These complexes enter the nucleus and bind the responsive elements of the target genes for their activation. The centrality of this regulatory pathway was confirmed by the finding that hemojuvelin (HJV), which is mutated in severe juvenile hemochromatosis (Papanikolaou et al., 2004), acts as a co-receptor of the BMP/SMAD pathway in the liver (Babitt et al., 2006). The 20 different members of the BMP family are produced by various cell types and have different functions, including osteogenesis, embryogenesis, and differentiation. Initial experiments used the osteogenic BMP2 and BMP4 to stimulate hepcidin expression in cultured hepatic cells (Truksa et al., 2006). In vitro studies showed that also BMP5, 7 and 9 can induce SMAD pathway and hepcidin expression in primary hepatocytes (Truksa et al., 2006) but after the finding that BMP6 is modulated by systemic iron and, more important, that BMP6^-/- mice suffer of severe iron overload and the lack of liver hepcidin it was accepted that BMP6 is the major regulator of hepcidin expression (Andriopoulos et al., 2009; Meynard et al., 2009). The dimers of type-II and type-I BMP-receptor participate in BMP/SMAD signaling together with various co-receptors and inhibitors. In the hepatic signaling, ALK2/ALK3 are the predominant BMPR type-I, and ActRIIA is the predominant type-II (Xia et al., 2008) and, of note, the GPI-anchor protein HJV acts as an essential co-receptor for hepcidin expression (Babitt et al., 2006). HJV is a member of the repulsive guidance molecule (RGM) family, which includes RGMa and DRAGON (RGMb), GPI-anchored proteins apparently involved in BMP signaling in different tissues (Corradini et al., 2009). HJV is expressed in skeletal and heart muscle and particularly in the liver where acts as an essential regulator of the signaling. It is also processed by the convertase furin into a soluble form that may act as a decoy and reduce hepcidin expression (Kuninger et al., 2008; Silvestri et al., 2008). It is degraded by the liver-specific serine protease Matriptase-2 (MT2, alias TMPRSS6; Silvestri et al., 2009). Inactivation of MT2 in mice and in human results in hepcidin increase and causes iron refractory iron deficiency anemia (IRIDA; Du et al., 2008; Finberg et al., 2008; Folgueras et al., 2008). Neogenin is another transmembrane protein involved in the signaling. It is ubiquitously expressed and interacts with RGM proteins. In the liver it interacts with both HJV and MT2 and may be required for the proper assembly of HJV-BMP ligand-BMPR-I/BMPR-II complex to initiate the BMP signaling and induce hepcidin expression (Zhang et al., 2005; Enns et al., 2012). Neogenin may also form a ternary complex with MT2 and HJV that facilitates HJV cleavage by MT2. Thus Neogenin can stimulate or suppress the BMP signaling by favoring the assembly of BMPs/HJV/BMPR complex or by facilitating MT2 activity, respectively (Zhao et al., 2013). A point not yet fully elucidated regards the involvement of HFE and TFR2. These genes are mutated in type I and III hemochromatosis, both characterized by relatively low hepcidin levels. HFE can bind both TFR1 and TFR2 and a model has been proposed in which HFE and TFR2 act as a complex and contribute to the complete activation of BMP/SMAD pathway (Gao et al., 2010). Other studies indicated that HFE and TFR2 are involved in the activation of MAPK, a pathway that cross talks with the BMP/SMAD and contribute to hepcidin expression (Poli et al., 2010; Figure 2A).

Regulation of the BMP/SMAD signaling occurs also after the phosphorylation of SMAD1/5/8 and ensures fine tuning at cytosolic level. SMAD7 antagonizes the recruitment of SMAD4 and the translocation of SMAD1/5/8-SMAD4 complex into the nucleus and recently it has been demonstrated that in hepcidin promoter there is a SMAD7-binding motif for a direct inhibition of the promoter (Mleczko-Sanecka et al., 2010).

Another well characterized pathway of hepcidin regulation involves the inflammatory cytokine IL6 that binds its specific receptor and activates JAK1/2 to phosphorylate STAT3. The phosphoSTAT3 translocates into the nucleus and binds the STAT3 response element (STAT3-RE) in hepcidin promoter, stimulating its transcription (Verga Falzacappa et al., 2007). This pathway can be activated by other cytokines including IL22 and Oncostatin-M which also increase hepcidin transcription, (Chung et al., 2010; Armitage et al., 2011). This inflammatory signaling relies on the BMPs/SMAD pathway to trigger hepcidin expression; in fact SMAD4-KO mice do not respond to the stimulation of IL6 (Wang et al., 2005). Activin-B, which belongs to the TGF-beta family, was shown to be stimulated by inflammation and to induce hepcidin via the BMP/SMAD pathway, thus it may cooperate and enhance the IL6-dependent stimulation and it could represent a connecting component between iron status and inflammation (Besson-Fournier et al., 2012; Figure 2B).

The only known receptor for hepcidin is ferroportin, which is the sole cellular iron exporter. Ferroportin (FPN) is expressed by enterocytes of the duodenum, by macrophages that process effete RBC and by liver, and is responsible for the release of iron to transferrin, in a mechanism that needs the assistance of a copper ferroxidase such ceruloplasmin or haephestin (Nemeth et al., 2004; Hentze et al., 2010). The iron export activity of ferroportin is essential for the regulation of systemic iron homeostasis, but its biochemical properties and the mechanism of iron transport are not well characterized. It is a 62.5 kDa protein with 12 transmembrane domains, the N-terminus and possibly also the C-terminus are cytosolic (Liu et al., 2005; Figure 3A). Mutation in FPN gene lead to type IV hemochromatosis (ferroportin disease), which is dominantly transmitted. This has suggested that ferroportin forms functional dimers (De Domenico et al., 2005), but direct studies indicated that it is a monomer (Montosi et al., 2001; Liu et al., 2005; Wallace et al., 2010; Figure 3B). The ferroportin mRNA has an IRE on the 5′UTR that may be responsible for its iron-dependent regulation (Muckenthaler, 2008). However, the major regulatory step occurs at a post-translational level caused by hepcidin (Nemeth et al., 2004). In fact the binding of hepcidin causes ferroportin internalization and degradation, with the effect to reduce systemic iron availability (Ganz and Nemeth, 2012). Further studies showed that Hepcidin-25 binds an extracellular loop of FPN adjacent to the cytosolic loop containing the two tyrosines required to signal internalization (Nemeth et al., 2006). The binding involves disulfide bridging with FPN Cys326 (Fernandes et al., 2009; Figure 3A), ferroportin ubiquitination prevalently at the level of lysines present in a third cytoplasmic loop of ferroportin (possibly Lys 229 and 269), and proteasomal degradation (Qiao et al., 2012; Figure 3B).

Liver is the major producer of hepcidin, and the fine tuning of its expression sets the level of circulating hepcidin to govern systemic iron homeostasis. Various other tissues express hepcidin mRNA and protein suggesting the presence of autocrine or paracrine circuits that may contribute to the regulation of local iron distribution. This may be particularly important in the central nervous system (CNS) that is separated from the rest of the body. The blood–brain barrier (BBB) and the blood/CSF barrier are the major sites of iron exchange with the periphery (Rouault et al., 2009; Figure 4A). These barriers act as semipermeable cellular gates characterized by tight junction and specialized transcellular carriers mediating influx or efflux. Transferrin and its receptor take part in most of iron transfer, but the details on the regulatory mechanism are missing. Of interest is that cells of the blood/CSF barrier express proteins of the hepcidin/ferroportin axis (Figure 4B). The imaging techniques and histology of the different areas of the brain showed that regions with neurodegeneration exhibit also iron accumulation in pathological conditions such as Alzheimer’s disease (AD; Bishop et al., 2002), Parkinson’s disease (PD; Berg et al., 2001; Zecca et al., 2004), and neurodegeneration with brain iron accumulation (NBIA; Hayflick, 2006), while restless leg syndrome (RLS) is associated with reduced CNS iron content (Clardy et al., 2006). There is evidence that the hepcidin/ferroportin axis might play a role in the iron decompartmentalization occurring in these disorders. In fact, not only hepcidin and ferroportin, but also the accessory proteins in iron transfer ceruloplasmin, hephaestin and DMT1, and those involved in hepcidin regulation (BMPs, IL6, Tfr2) are expressed and regulated in the brain (Figure 4B). Hepcidin and ferroportin mRNA and protein were detected in different murine and human cerebral areas (Clardy et al., 2006; Zechel et al., 2006) and hepcidin intraventricular injection resulted in downregulation of FPN protein (Wang et al., 2010b). Systemic iron inflammation or acute iron overload induced a significant increase in cerebral hepcidin expression in mice and rats (Malik et al., 2011; Wang et al., 2011; Sun et al., 2012) thus suggesting that the machinery controlling hepcidin expression in the brain is similar to the hepatic one. Novel evidence suggests that perturbation of the cerebral hepcidin-FPN axis may contribute to local deregulation of iron homeostasis. They include an increase of hepcidin in the brain in a murine model of cerebral ischemia (Ding et al., 2011), and a reduction of both ferroportin and hepcidin detected in lysates obtained from AD patients (Raha et al., 2013). Furthermore, calorie restriction prevented both the increase in cerebral hepcidin mRNA and the impairment of learning and memory observed in an experimental model of aging (Wei et al., 2014). Further characterization of the machinery controlling iron balance in the brain is needed, but attention should be given at the possible modulation of hepcidin expression in CNS caused by pharmacological intervention aimed at regulating systemic iron homeostasis, in particular those involving molecules that can pass the BBB.

Hepcidin is the hormone of iron and inflammation and its deregulation occurs in all iron related disorders, including the ones characterized by iron restriction and anemia in which hepcidin is abnormal (Ganz and Nemeth, 2011). Elevated hepcidin levels are associated with secondary iron overload, genetic IRIDA, chronic infectious and inflammatory diseases resulting in anemia of inflammation (AI). The finding that hepcidin is upregulated by the inflammatory cytokine IL6 (Verga Falzacappa et al., 2007) contributed to explain the anemia of chronic diseases (ACD) alias AI. This occurs in a variety of disorders, like infections, chronic kidney diseases (CKDs), rheumatoid arthritis, and cancer (Wang et al., 2012) including multiple myeloma (Maes et al., 2010), a severe malignant disorder of plasma cells. Currently ACD therapy includes erythropoiesis-stimulating agents and intravenous iron (Goodnough et al., 2010), which may have adverse effects and are scarcely effective (Glaspy, 2012; Fung and Nemeth, 2013). For example inflammation often induces erythropoietin resistance (Macdougall and Cooper, 2002). Alternative treatments have been proposed and hepcidin antagonists seem to be the best candidates to treat these disorders (Wang et al., 2012).

The mechanisms involved in the regulation of hepcidin expression are complex and partially known, and the approaches can use different targets to downregulate hepcidin or its function, as described in recent reviews (Sun et al., 2011; Fung and Nemeth, 2013).

The hepcidin antagonists are expected to find clinical use mainly for the treatment of inflammatory anemia which, although widely diffused in clinical practice, has few animal models with different properties, as recently reviewed (Rivera and Ganz, 2009). Here is a short description of the ones so far described in past and recent papers focusing only on the well-known model.

This review shows that many laboratories are studying different pharmacological means to neutralize hepcidin expression or activity in order to cure inflammatory anemia. They produced a number of promising approaches, and some of them have been tested in animal models. Most of them seemed to be effective in reducing hepcidin expression or activity under acute conditions, but it is still unclear if and how they are efficient in the treatment of anemia. One of the problems is the lack of adequate animal models for inflammatory anemia, as indicated above. Mice models are rather complex, and rat models seems to mimic more closely the human disease, but the absence of transgenic rats for hepcidin and inflammatory cytokines does not allow a detailed characterization. Monkeys have been used to induce inflammatory response, but not anemia (Cooke et al., 2013). The described antagonists (Table 1) originate from different and novel biotechnological techniques, including humanized anti-hepcidin antibodies, aptamers, anticalin, siRNAs, and the old traditional heparin. Some of them are in clinical trials, and perhaps in a few years we will know if the downregulation of hepcidin really meets the expectation to improve the anemia in most, or some chronic diseases. All the antagonists have some advantages and problems. For example the humanized antibodies have a long half-life in vivo, but their production is highly expensive. Aptamer and anticalin inactivate hepcidin, but their fate is unclear. The specific and efficient delivery of siRNA is complex. Non-anticoagulant heparins are probably the best known, most convenient and safer agents. After 70 or more years of use in clinic, most of problems and side effects of heparins are known. They include thrombocytopenia, elevation in serum aminotransferase, hyperkalemia, alopecia and osteoporosis, but they occur rarely and are transient. The removal of anticoagulant activity has been resolved and the clinical trials of these agents as hepcidin antagonist should not be far away. Also the negative effects of treatments for hepcidin inhibition should be taken into account. They are expected to increase systemic iron availability and absorption, which may favor iron-dependent oxidative damage in some parenchymal tissues. If the pharmacological agents are capable to cross BBB and enter the brain, they may alter CNS iron homeostasis with unpredictable effects. In addition, hepcidin is known to have an antimicrobial activity, which is considered low, but its biological role needs to be established.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.