BMP: Bone Morphogenic Protein; BMPRIIA: BMP Receptor Type IIA; BMPRI: BMP Receptor Type I; ALK 2/3: ALK, Activin-Like Kinase; Endofin: Endosome-associated FYVE-domain; SMAD1/5/8: Sma and Mothers Against Decapentaplegic Homolog 1/5/8; SMAD4: Sma and Mothers Against Decapentaplegic Homolog 4; ATOH8: Atonal Homolog 8; BMPRE: BMP Response Elements; HFE: Hemochromatosis Protein; HJV: Hemojuvelin; IL6: Interleukin 6; PARP-1: Poly ADP Ribose Polymerase 1; TMPRSS6: Transmembrane Protease Serine 6; Tf: Transferrin; TfR2: Transferrin Receptor 2

Iron is an important mineral for most life on Earth, including humans. It is required as cofactor for a number of proteins including cytochromes and plays a major role in oxygen transport by erythrocyte hemoglobin and oxygen storage by muscle myoglobin. Just as iron deficiency is detrimental, iron overload is also deleterious as there is no specific pathway to excrete iron. Therefore, iron is strictly regulated both at the cellular and systemic levels. Its absorption by intestinal enterocytes and concentration in serum is strictly regulated by liver produced hormone, hepcidin in association with iron exporter, ferroportin. Hepcidin expression in response to body iron level is primarily regulated via Bone Morphogenetic Protein/Sma and Mothers against Decapentaplegic (BMP/SMAD) pathway, although other signaling pathways may exist [1]. This mini review summarizes present day knowledge of the key players involved in iron absorption, transport, storage, and homeostasis. It also synopsizes regulation of hepcidin by known major pathways.

As a vital mineral, iron concentration is strictly maintained in circulation within 10-30 microM to ensure adequate supply for biosynthesis of hemoglobin (erythropoiesis), myoglobin and cytochromes, and for other essential metabolic functions. However, its concentration is kept at low to prevent generation of free radicals [2] in the presence of H₂O₂ that can cause damages to cellular components. It is virtually insoluble under physiological condition, so animals have developed specialized molecules for its acquisition, transport, and storage in a soluble and nontoxic form. Another set of molecules evolved for its intracellular and systemic regulation.

Under steady state, very little iron is absorbed or lost. Most of the iron requirement is met by recycling from erythrocyte turn-over. Senescent erythrocytes are catabolized by macrophages in spleen, liver and reticuloendothelial system, and the released iron is used by bone marrow reticulocytes for synthesis of hemoglobin. There is no specific excretory pathway for iron, although small amount (1-2 mg) is lost from the body through a variety of ways including urination, defecation, sweating, and exfoliating old/matured cells [3]. Thus, vast majority of body iron is recycled into hemoglobin of circulating erythrocytes (2500 mg in human) and bone marrow erythroid precursors (300 mg), and the excess is stored in the liver (1000 mg) and splenic macrophages (600 mg). Hepatocytes serve as the important reservoir that stores or releases iron to maintain homeostasis. Under normal condition, a small amount of iron is found in circulating plasma.

Dietary ferric (Fe³⁺) iron is absorbed by mature enterocytes-cells of intestinal epithelia, following reduction to ferrous (Fe²⁺) in the intestinal lumen by ferric reductases. Two main pathways to absorb iron from the lumen are associated with HCP1 (heme carrier protein 1) for inorganic non-heme iron and DMT1 (divalent metal transporter 1) for organic heme iron. They are expressed on the enterocyte apical membrane and transfer Fe²⁺ ions into its cytoplasm [4].

Absorbed dietary iron in enterocytes (and stored iron in hepatocytes and macrophages) is secreted into plasma via iron exporter ferroportin [5], after being re-oxidized to Fe³⁺ by ferroxidases, ceruloplasmin and hephaestin [6]. Once in plasma, Fe³⁺ binds tightly, but reversibly Tf, a plasma carrier protein highly expressed and actively secreted by hepatocytes. Cells acquire iron from circulating Fe³⁺ bound Tf via transferrin receptor 1 (TfR1) localized on the cell membrane through endocytosis [7]. Ferric iron is released from endosomes carrying Fe³⁺-Tf-TfR1 ternary complex and undergoes reduction to Fe²⁺ by reductases.

Excess iron in hepatocytes is stored in complex form with cytoplasmic iron-storage protein ferritin. Within its ferooxidase site, Fe²⁺ is oxidized with dioxygen [8,9] to ferric Fe³⁺ ion, and stored in solid, nanoparticular, and mineral form [10]. This limits radical production via Fenton reaction between ferrous iron and hydrogen peroxide. When needed, ferritin is degraded by lysosomes [11] and the stored Fe³⁺ is reduced for its exit [12] and transit to the exporter ferroportin. It is not known how this reduction and transit to ferroportin is carried out, but a ferrireductase may be involved. Copper-containing ferroxidases ceruloplasmin, hephaestin, and possibly Zyklopen [13] use molecular oxygen to re-oxidize ferroportin bound Fe²⁺ to Fe³⁺ before being taken up by Tf. Thus, both ferritin and Tf keep iron in unreactive form and prevent formation of oxygen radicals and cellular damage [14].

Dietary iron is absorbed by enterocytes to replace iron losses from the body. Cellular iron uptake, storage and export have to be coordinately regulated in the body. Cells monitor iron concentration through a feedback control mechanism involving cytosolic ironregulatory proteins 1 and 2 (IRP1/2). This regulation is exerted via posttranscriptional mechanism utilizing Iron Responsive Elements (IREs) located in the untranslated region (UTR) of mRNAs [15] of proteins involved in iron metabolism. IRE is present in the 5' UTR of ferritin and ferroportin mRNA, and 3' UTR of TfR1 and DMT1 mRNAs. With high iron concentration, IRPs lose affinity for IREs resulting in increased translation of 5′ IRE-containing mRNAs and degradation of the 3′ IRE-containing mRNAs [15]. Thus, precisely regulated control by IRPs allows cells to acquire enough iron for their metabolism without reaching toxic concentration.

In addition to intracellular control, iron homeostasis is maintained at the systemic level by controlling absorption through intestinal enterocytes and export from body stores. Key to this maintenance is a 25-amino-acid peptide hormone, hepcidin (HAMP gene product) produced predominantly by hepatocytes and secreted into circulation [16]. Secreted hepcidin in serum is transported in complex with beta 2-macroglobulin [17]. Hepcidin negatively regulates absorption of dietary iron and release from stored sources into circulation [18] through binding to the exporter ferroportin and leading to its degradation. Thus, high serum hepcidin level decreased iron absorption by enterocytes as well as iron export from hepatocytes and macrophages leading to reduced iron entry into circulation and intracellular retention [19,20]. Sustained elevation of hepcidin in circulation, as occurs in many inflammations, leads to anemia due to reduced availability of iron for erythropoiesis, a condition known as anemia of inflammation. On the other hand, below normal hepcidin concentration accounts for increased dietary iron absorption and tissue iron overload, leading to iron overload disease, hemochromatosis. This disease is associated with disrupted control of dietary iron absorption and progressive iron overload in tissues leading to multiple organ damage and failure in humans. The most frequent type is hereditary hemochromatosis (HH), developed by chronic hyper-absorption of dietary iron in individuals with mutations in the HFE gene [21]. HFE encodes an atypical major histocompatibility complex (MHC) class I protein, however, unlike other members of MHC class I, it does not possess peptide antigen presentation. The majority of HH patients carry a C282Y mutation that abolishes a disulfide bridge in the HFE protein, preventing its association with β2- microglobulin for proper processing, transport to the membrane and cell surface expression [22]. Unprocessed C282Y mutant protein is degraded by proteasome in the cells [23]. Mutations in a number of other genes such as TfR2, homojuvelin (HJV), and bone morphogenetic protein 6 (BMP6) lead to low hepcidin level and develop HH. In fact, HAMP1 regulation is more severely impaired in Bmp6^-/- and Hjv^-/- mice than in Hfe^-/- and Tfr2^-/-mice.

Current knowledge of hepcidin regulation appears to occur at the transcriptional level. Two major stimuli that regulate hepcidin promoter activity are body iron status and inflammation: hepcidin transcription in liver is induced by serum iron level or inflammation, and is inhibited by iron deficiency, increased erythropoietic drive or hypoxia [24,25].

Iron-mediated regulation

BMP/SMAD pathway: Iron induced hepcidin activation is mainly regulated through BMP (bone morphogenetic protein) signaling pathway [1]. BMP6 binds as a ligand to BMP receptors (BMPR) type I and II, promoting phosphorylation of downstream BMP mediators such as SMAD1, SMAD5 and SMAD8 (SMAD1/5/8=SMA and mothers against decapentaplegic homolog 1, 5 or 8). Among BMPR type I, only ALK2 and ALK3 are expressed in human [26] and murine [27] hepatocytes. As for BMPR type II, hepatocytes express only ActRIIA [26]. Transgenic mice with liver specific deletion of Alk2 or Alk3 [27] showed impaired iron induced expression of hepcidin and iron overload.

The phosphorylated SMAD1/5/8 associates with cytoplasmic SMAD4 and translocates to the nucleus, where the complex activates hepcidin transcription, in addition to other target genes, upon binding to BMP responsive elements (BMP-RE1 and BMP-RE2) at proximal and distal sites of HAMP promoter [28]. Mice with liver-specific ablation of Smad4 had low plasma hepcidin level, and hepatocytes with knocked down SMAD4 do not respond to iron overload and BMP signaling [29], supporting an important role of SMAD4 in ironmediated hepcidin expression.

BMP signaling pathway is the critical iron-mediated regulator of hepcidin as BMP6, unlike other members of BMP protein family, is responsive to iron concentrations. BMP6 is produced in nonparenchymal cells of the liver and activated by iron; its expression is proportional to hepatic iron level [30]. Transgenic mice with ablated BMP6 lost the ability to activate hepcidin in response to iron overload, which can be rescued by only BMP6 [31,32]. In addition, neutralizing BMP6 antibody can decrease hepcidin expression and trigger an increase in serum iron concentrations in mice [32]. These data suggest a unique, perhaps central, role of BMP6 in hepcidin transcriptional regulation in response to iron level.

BMP signaling in liver requires BMP co-receptor hemojuvelin (HJV), since it forms complex with BMP receptors to enhance downstream signaling. Mutations in HJV cause decreased BMP signaling, resulting in decreased hepcidin expression, followed by severe iron overload [33] and HH. Consistent with this, cleavage of HJV by liver expressed, membrane serine-threonine protease, matriptase-2 encoded by TMPRSS6 results in suppressed signaling through the BMP pathway, and consequently, decreased hepcidin expression [34]. Tmprss6^-/- mice have increased HAMP1 expression, decreased ferroportin level and severe iron-deficient anemia. Mutations in TMPRSS6 produce iron-refractory iron-deficiency anemia in humans [35]. Evidences suggest that TMPRSS6 expression is positively correlated by iron level through BMP6 that requires intact BMP/SMAD pathway and BMP6 mediated induction of the transcription factor, Id1 [36]. In contrast, a recent result showed low iron level increased TMPRSS6 expression in hepatocyte cell culture [37]. In any case, matriptase-2 serves as a negative regulator of hepcidin activation by the BMP pathway.

Besides matriptase-2 , other important modulators of the BMP/ SMAD pathway include endofin, ATOH8 and SMAD7. Endosomeassociated FYVE-domain protein, endofin anchors SMAD1/5/8 proteins to BMP receptors and enhances SMADs phosphorylation, and thus, activates BMP signaling [38]. Using Endofin specific siRNA in hepatic cultured cells, Goh et al. [39] showed that endofin is required for BMP6 induced hepcidin activation as it facilitates SMAD1/5/8 phosphorylation.

Transcription factor ATOH8 also activates hepcidin expression by enhancing SMAD1/5/8 phosphorylation, although this activation in HEK 293 cells is also suggested by direct binding to E-boxes of hepcidin proximal promoter [40]. Atoh8 contributes to hepcidin sensitivity to iron as it is correlated with plasma iron and erythroid activity in mice [40,41]

Activation of inhibitory SMAD7, which regulates BMP pathway by negative feedback loop, decreases hepcidin transcription [42]. Evidences suggest SMAD7 directly suppresses hepcidin expression as specific siRNA resulted in increased HAMP transcription. In mice hepatocytes, overexpression of SMAD7 suppresses HAMP1 expression and prevents HAMP1 induction by BMP6.

Pathways involving HFE: Although evidences described above clearly place MBP6 at the center of sensing iron level and BMP/SMAD as the central pathway of hepcidin regulation in response to iron, alternative model of hepcidin regulation emerged through sensing iron level via the interaction of Fe³⁺-Tf with its receptors. Studies in Hfe^-/- mice showed that even with iron overload and corresponding increase in BMP6 mRNA, no increase in phosphorylation level of SMAD1/5/8 or HAMP1 transcription was observed. In consistent, despite increase in BMP6 mRNA, phosphorylation of SMAD1/5/8 and expression of HAMP are inappropriately low in individuals with HH carrying C282Y mutations. No change in SMAD4 mRNA level is noticed. Interestingly, inhibitory Smads, SMAD7 and SMAD6, are upregulated in these patients. All these suggest existence of additional pathways for HAMP regulation.

The first pathway involving HFE, reported by Bennet et al. [43], demonstrated that HFE binds both transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2) [44] on the cell membrane. However, the choice between TfR1 and TfR2 is determined by concentration of Fe³⁺- Tf: increased concentration disrupts HFE-TfR1 interaction and shifts HFE binding towards TfR2. HFE-TfR2 complex, in turn, activates hepcidin transcription [45,46]. Mutated HFE fails to form this complex with TfR2 and suppresses hepcidin level. The mechanism of this regulation is still unclear, although both HFE and TfR2 probably contribute to hepcidin up regulation through BMP/SMAD signaling pathway in response to increased iron [31,47]. The role of hepatic Tfr2 as a regulator of hepcidin transcription has been confirmed in transgenic mice studies. Low hepcidin level and iron overload in liver have been found in Tfr2 null mice as well as in liver specific Tfr2 knockout mice model [48,49]. Thus, in this pathway, HFE utilizes its interaction with transferrin receptors on the cell membrane to regulate hepcidin expression upon changes in iron bound transferrin concentration in serum.

The second pathway of hepcidin regulation by HFE has been recently reported by Wu et al. [50]. They demonstrated that HFE regulates hepcidin transcription via BMP/SMAD pathway. BMP6 phosphorylates BMPR type I Alk2 and/or Alk3 that are expressed in the liver and critical for hepcidin transcription [27]. Analysis of HH patients with mutated HFE and HH model mice showed impaired BMP signaling suggesting critical role of HFE in BMP/SMAD mediated hepcidin regulation [51]. Wu et al. [50] determined that Hfe binding to hepatic Alk3 reduces its degradation by proteasome and activates hepcidin expression, thus linking HFE to BMP/SMAD pathway. Consistent with this, Hfe deficient mice show reduced Alk3 protein level and, consequently, reduced hepcidin expression. This is the first evidence of hepcidin transcription activation by HFE via BMP signaling.

Our results in cultured cells showed that PARP1 (poly ADP ribose polymerase 1), a nuclear protein, strongly represses HFE expression via binding to a cruciform structure in the distal HFE promoter. PARP1 specific siRNA increases HFE mRNA and protein. Likewise, PARP1 degradation, induced by iron containing molecules such as hemin and FeCl₃, increases HFE expression by releasing the repression [52]. Other studies demonstrated that PARP1 disrupts SMAD complex assembled on DNA target sites by ADP-ribosylating (PARylating) Smad4 and attenuated SMAD mediated response [53]. PARylation of SMAD proteins by PARP1 is a key step in controlling the strength and duration of SMAD mediated transcription [53,54]. Thus, PARP1 negatively regulates transcription of SMAD target genes. Taken together, PARP1 can reduce Hepcidin expression in two ways: via PARylation of SMAD4 assembled on BMPRE1 and 2 sites in BMP/ SMAD mediated pathway and/or via reducing HFE expression in pathways requiring HFE. Therefore, degradation of PARP1 in response to high iron can apparently release these repressions on hepcidin transcription. Likewise, PARylation activity inhibitors can increase hepcidin expression in BMP/SMAD pathway. It will be interesting to investigate if PARP1 inhibitors can alleviate iron overload symptoms in HH mice.

Inflammation mediated regulation

The second major signaling pathway known to regulate hepcidin transcription is the JAK (Janus kinase)–STAT3 (signal transducer and activator of transcription 3) pathway; the stimulus for this regulation comes mainly through inflammatory cytokines such as interleukin-6 (IL-6). IL-6, released upon inflammation, binds to its receptors on the cell membrane leading to activation of JAK which phosphorylates predominantly STAT3 protein in hepatocytes. Phosphorylated STAT3 translocates to the nucleus and binds to a STAT-specific site in target gene promoters. It directly regulates hepcidin through a STAT binding site in proximal region of its promoter [55-57]. IL-6 also binds BMP receptor Alk3 and induces hepcidin expression in mice [58], suggesting a cross-talk exists between BMP and IL-6 regulation. Another mechanism of hepcidin up-regulation upon inflammation involves activin B, a member of TGF-beta protein superfamily that includes BMP6 [59]. Activin B binds to BMP receptor type I, ALK3 to activate hepcidin transcription in hepatocytes via BMP/SMAD pathway (Figure 1) [60-65].

Figure 1: Overview of Hepcidin transcription regulation in hepatocytes [In response to high body iron, in primary pathway BMP6 binds activated BMP receptor complex, consisting of BMPRIIA (Act RII) and BMPRI (ALK) and co-receptor HJV, which phosphorylates SMAD1/5/8 anchored by endorphin protein. ATOH8 enhances this phosphorylation, while matriptase2 encoded by TMPRSS6 attenuates this phosphorylation via proteolytic cleavage of HJV. The phospho-SMAD1/5/8 recruits the common SMAD4 to form a transcription complex, which upon transport to the nucleus, binds BMP response elements (BMPREI and II) on the hepcidin promoter and upregulates hepcidin transcription. In pathways involving HFE, complexes are formed between HFE with either holotransferrin bound transferrin receptor 2 (2Fe³⁺-Tf-TfR2) or ALK and transmit signal via BMP or other pathway-the mechanisms are not fully characterized. Inflammation induced pathway through IL6 and JAK/STAT3 is also shown. Evidence shows that PARP1 represses HFE directly via binding to HFE promoter, while HAMP indirectly via dissociation of SMAD4- SMAD1/5/8 complex through PARylation of SMAD4. Iron induced breakdown of PARP1 releases the repression on HFE promoter. Both iron and PARP1 activity inhibitors thus can remove PARP1 inhibitory activity on HAMP promoter].

Both iron deficiency and iron overload are deleterious. Thus, body iron levels are strictly maintained by regulating absorption of dietary iron and release of stored iron by the hepatic hormone, hepcidin, its impaired regulation is associated with all iron related disorders. Over the past two decades, an incredible amount of progress has been made in our understanding of iron homeostasis and hepcidin regulation. Hepcidin expression is tightly regulated at transcription level primarily via BMP/SMAD pathway. BMP6 produced in non-parenchymal cells of liver is the predominant ligand and main upstream positive regulator of hepcidin responding to iron level.

However, several key questions still remain. For example, it is still not clear how BMP6 sense iron in the non-parenchymal cells and what other molecules are involved. What are the DNA binding and transcription partners that drive transcription of HAMP? How iron is sensed in HFE-ALK3 pathway? Since inhibitory SMAD7 is upregulated in HFE deficient mice and individuals, is it the role of HFE to keep in check the inhibitory pathway? Whether PARP1 plays a significant role in HAMP regulation and its inhibitors can alleviate iron overload symptoms in HH mice. The answers to these questions will provide further insight into hepcidin mediated iron homeostasis.