Juvenile Hemochromatosis (JH) is an autosomal recessive disorder caused by a failure to prevent excess iron entry into the bloodstream, and characterized by progressive tissue iron overload (Pietrangelo, 2010). Although iron's redox properties are critical for its role in many fundamental biological processes from cellular respiration to oxygen transport, iron excess can lead to toxic free radical generation. If left untreated, JH patients develop multiorgan dysfunction as a consequence of iron overload, including cirrhosis, cardiomyopathy, diabetes mellitus, and hypogonadotrophic hypogonadism, before the age of 30 (Pietrangelo, 2010).

The identification of hepcidin as a master regulator of systemic iron balance was a major advance in understanding the pathophysiology of JH (Ganz, 2013). A defensin-like peptide produced predominantly by hepatocytes, hepcidin controls iron entry into the bloodstream from dietary sources, recycled red blood cells, and body storage sites by inducing degradation of the iron exporter ferroportin (Ganz, 2013). Hepcidin expression is stimulated by iron and inflammation to limit iron availability, while hepcidin is inhibited by iron deficiency, anemia, and hypoxia to increase iron availability for erythropoiesis (Babitt and Lin, 2010; Ganz, 2013). Hepcidin deficiency is the common pathogenic mechanism underlying both adult and juvenile-onset hemochromatosis and contributes to the pathogenesis of iron loading anemias such as thalassemia, while its overproduction causes anemia of inflammation and iron refractory iron deficiency anemia (IRIDA) (Ganz, 2013). JH is caused by mutations in the gene encoding hepcidin itself (HAMP) or, more commonly, hemojuvelin (HJV, also known as HFE2 or RGMC) (Roetto et al., 2003; Papanikolaou et al., 2004).

HJV encodes a glycophosphatidylinositol (GPI)-linked membrane protein that is a member of the repulsive guidance molecule (RGM) family (Monnier et al., 2002; Samad et al., 2004). Currently, there are 43 identified HJV mutations that cause JH, with G320V being the most frequent (Table 1). HJV is expressed in the liver, and JH patients with HJV mutations and Hjv knockout mice exhibit significantly reduced hepatic hepcidin expression, thereby implicating HJV in the regulation of hepcidin synthesis (Papanikolaou et al., 2004; Huang et al., 2005; Niederkofler et al., 2005).

A breakthrough in understanding the mechanism of action of HJV in hepcidin regulation came when HJV was discovered to function as a co-receptor for the bone morphogenetic protein (BMP) signaling pathway (Babitt et al., 2006), analogous to its RGM family homologs (Babitt et al., 2005; Samad et al., 2005). Importantly, this BMP signaling function of HJV was demonstrated to be crucial for its role in regulating hepcidin expression (Babitt et al., 2006) (Figure 1).

BMPs belong to the Transforming Growth Factor-beta (TGF-β) superfamily of ligands (Shi and Massagué, 2003). In the canonical signaling pathway, BMP ligands bind to type I and type II serine threonine kinase receptors to induce phosphorylation of cytoplasmic SMAD1, SMAD5, and SMAD8 proteins. These SMAD proteins form a complex with SMAD4 and translocate to the nucleus to regulate gene transcription. This signaling pathway is further regulated at multiple levels in order to generate a precise signal in a specific cellular context (Shi and Massagué, 2003).

HJV and other RGM family members function as BMP co-receptors that bind selectively to BMP ligands and receptors to enhance SMAD phosphorylation in response to BMP signals (Babitt et al., 2005, 2006; Samad et al., 2005). All RGMs share the ability to bind to the BMP2/BMP4 subfamily and enhance BMP2/BMP4 signaling (Babitt et al., 2005, 2006; Samad et al., 2005; Wu et al., 2012). Moreover, all RGMs utilize BMP type I receptors ALK2, ALK3, and ALK6, and allow preferential signaling through the BMP type II receptor ACTRIIA (Xia et al., 2007, 2008, 2010). However, HJV is unique from other RGMs in that it exhibits preferential ability to bind to the BMP5/BMP6/BMP7 subfamily compared with RGMA and RGMB (Wu et al., 2012).

The BMP-HJV-SMAD signaling pathway activates hepcidin transcription directly through specific BMP-responsive elements (BMP-REs) on the hepcidin promoter (Casanovas et al., 2009; Truksa et al., 2009a). A mutation in the proximal BMP-RE was associated with a more severe iron overload phenotype in a patient with classical HFE hemochromatosis, demonstrating its importance in hepcidin regulation in humans (Island et al., 2009). In mice, liver-specific disruption of Smad4, the BMP receptors type I Alk2 or Alk3, or the ligand Bmp6 result in hepcidin deficiency and iron overload, supporting the important role of these specific BMP-SMAD pathway components, in conjunction with HJV, in hepcidin regulation in vivo (Wang et al., 2005; Andriopoulos et al., 2009; Meynard et al., 2009; Steinbicker et al., 2011a).

In addition to the GPI-anchored membrane form of HJV, endogenous soluble HJV (sHJV) protein is detectable in human and rodent serum. (Lin et al., 2005; Zhang et al., 2007; Chen et al., 2013). Multiple mechanisms have been proposed for endogenous sHJV generation, including cleavage by the pro-protein convertase furin and the type II transmembrane serine protease TMPRSS6 (Kuninger et al., 2008; Lin et al., 2008; Silvestri et al., 2008a,b). Whereas membrane HJV is a co-receptor for the BMP signaling complex (Babitt et al., 2006), sHJV can antagonize BMP signaling, presumably by binding and sequestering BMP ligands from interacting with cell-surface BMP type I and type II receptors (Babitt et al., 2007) (Figure 1). Indeed, the relative binding affinity of HJV for various BMP ligands roughly correlated with the ability of sHJV to inhibit their biological activity (Babitt et al., 2007; Wu et al., 2012).

Although exogenous sHJV inhibits BMP-SMAD signaling, the source, amount, and physiologic role(s) of endogenously produced sHJV in vivo are not well-understood. There is some evidence suggesting that endogenous sHJV is increased by iron deficiency and reduced by iron loading (Lin et al., 2005; Zhang et al., 2007; Silvestri et al., 2008a; Brasse-Lagnel et al., 2010; Chen et al., 2013). Interestingly, the furin cleaved form of sHJV appears to be more potent to inhibit BMP signaling and hepcidin compared with the TMPRSS6-cleaved form (Maxson et al., 2010). Whether HJV cleavage mainly represents a mechanism to remove the activating effects of liver membrane HJV, or whether endogenous sHJV has a direct BMP-SMAD inhibiting effect remains uncertain.

In addition to the liver, HJV mRNA is also highly expressed in skeletal muscle and heart (Niederkofler et al., 2004; Papanikolaou et al., 2004), and has been detected in other tissues (Rodriguez Martinez et al., 2004, Rodriguez et al., 2007; Gnana-Prakasam et al., 2009; Luciani et al., 2011). Tissue specific differences in HJV mRNA regulation and HJV protein glycosylation patterns have also been described (Niederkofler et al., 2005; Fujikura et al., 2011). It was previously hypothesized that skeletal muscle and/or heart could serve as a source of sHJV to suppress hepcidin synthesis in response to iron deficiency or hypoxia (Lin et al., 2005; Zhang et al., 2005). However, mice with a specific knockout of Hjv in skeletal ± cardiac muscle do not have altered hepcidin expression or systemic iron balance, at least under basal conditions or with dietary iron changes (Chen et al., 2011; Gkouvatsos et al., 2011). Whether strenuous exercise or hypoxia may uncover a role for muscle hemojuvelin remains uncertain. In contrast, hepatocyte specific Hjv knockout mice exhibit an iron overload phenotype similar to global Hjv knockout mice (Chen et al., 2011; Gkouvatsos et al., 2011). Thus, hepatic expression of HJV appears to have the most important physiologic role in systemic iron homeostasis regulation in vivo.

Iron regulates the activity of the BMP6-SMAD pathway to modulate hepcidin expression. Both circulating and liver iron appear to stimulate this pathway through different mechanisms (Ramos et al., 2011; Corradini et al., 2011a). In mice, liver iron content is positively correlated with liver Bmp6 mRNA levels and overall activity of the Smad signaling pathway (Kautz et al., 2008; Corradini et al., 2011a). Moreover, hepcidin induction by iron is inhibited by a neutralizing BMP6 antibody (Corradini et al., 2011a). These data suggest that liver iron modulates BMP6-SMAD signaling and hepcidin expression at least in part by regulating expression of BMP6 mRNA (Figure 1). It appears that liver iron regulates BMP6 expression mainly in nonparenchymal cells (Enns et al., 2013), and that iron loading in specific liver cell types may important for this regulation (Daba et al., 2013). However, the mechanism by which hepatic iron levels regulate BMP6 remains unknown. Notably, hepcidin is still increased to a lesser extent by chronic iron loading in Bmp6 and Hjv knockout mice, suggesting that these pathways do not completely account for hepcidin regulation by chronic iron loading (Ramos et al., 2011; Gkouvatsos et al., 2014).

Increases in circulating iron stimulate SMAD1/5/8 phosphorylation and hepcidin expression without affecting Bmp6 mRNA levels (Corradini et al., 2011a). How circulating iron activates SMAD1/5/8 phosphorylation is unknown, but may involve an interaction with other proteins that are mutated in adult-onset hereditary hemochromatosis (see section HFE and TFR2). HJV liver membrane protein expression itself does not appear to be regulated by iron (Krijt et al., 2012).

Iron administration and BMP6-SMAD signaling also up-regulate inhibitory SMAD7 and SMAD6, and TMPRSS6 (see section TMPRSS6), that can act as feedback inhibitors of BMP-SMAD signaling and hepcidin expression (Kautz et al., 2008; Mleczko-Sanecka et al., 2010; Meynard et al., 2011; Corradini et al., 2011a; Vujić Spasić et al., 2013). It has been hypothesized that these pathways may help prevent excessive hepcidin increases by iron to provide tight homeostatic control (Meynard et al., 2011; Corradini et al., 2011a).

Understanding the genetic basis for JH has yielded important insights into the molecular mechanisms of systemic iron homeostasis. Hepcidin and its receptor ferroportin are key regulators of body iron balance, and the BMP-SMAD pathway via the co-receptor HJV is a central regulator of hepcidin production (Figure 1). Knowledge of these pathways has already lead to the development of novel therapeutic strategies that target the molecular mechanisms underlying iron homeostasis disorders, with several new treatments currently being evaluated in human clinical trials (Fung and Nemeth, 2013). Future work will be needed to fully understand the mechanisms by which iron levels are sensed by the liver and integrated with other pathways to regulate BMP-SMAD signaling, hepcidin expression, and systemic iron homeostasis.