The regulated absorption of iron across the epithelium of the proximal small intestine is essential for maintaining body iron levels within a physiologically defined range. Over the last decade, the main intestinal iron transport proteins have been identified and focus has now turned to how they are regulated. The recognition that the liver-derived peptide hepcidin acts as a central regulator of body iron homeostasis, including iron absorption, has driven much of this work, and recent advances in the field will be highlighted in this review.
Iron transport across the intestinal enterocyte
Iron in the diet is usually considered to be in either the heme or nonheme form and both can be utilized by the intestinal epithelium (Fig. 1) . Nonheme iron crosses the apical brush border of enterocytes through the ferrous iron (Fe2+) transporter divalent metal ion transporter 1 (DMT1). As most dietary iron is in the ferric or Fe3+ state, it must first be reduced before it can be utilized, and a candidate iron reductase is the brush border protein duodenal cytochrome B (DcytB). Once within the enterocyte, iron has two basic fates depending on iron requirements. If iron demand is low, it will remain in the enterocyte sequestered by the iron storage protein ferritin and will be lost when the enterocytes are sloughed from the villus tip several days later. If iron is required by the body, it will cross the basolateral membrane through the iron export protein ferroportin (FPN) and enter the circulation in which it binds to plasma transferrin. The efflux of iron from enterocytes also requires the iron oxidase hephaestin. The mechanisms of heme iron absorption are less well defined. A candidate brush border heme transporter (heme carrier protein 1; HCP1) was recently described, but it was subsequently demonstrated that this protein transports folate far more efficiently than heme . Whether HCP1 is a bifunctional transporter that plays a physiological role in intestinal heme uptake or not has yet to be unequivocally demonstrated.
In recent years, the major contributions to our understanding of iron absorption have been centered around the pathways regulating intestinal iron transit and there have been few advances in elucidating the mechanism by which iron moves across the enterocyte per se. However, recent data on the function of the iron exporter FPN are potentially applicable to the gut. Ceruloplasmin (Cp) is involved in the release of iron from many cell types and appears to fulfil the same role in nongut tissues that hephaestin plays in the small intestine. De Domenico et al. [3•] have recently demonstrated that Cp plays a critical role in removing recently transported ferrous iron from FPN by facilitating its oxidation. In the absence of Cp, the iron remains bound to FPN and the protein-iron complex is targeted for degradation. How generalizable this phenomenon might be is not known, but hephaestin could potentially play a similar role in facilitating iron release from FPN on the basolateral membrane of enteroctyes.
Enterocyte iron homeostasis
The enterocyte iron transport machinery can respond to both external cues and variations in local iron concentrations. The former is essential for meeting body iron demands, such as the requirements of the erythroid marrow, whereas the latter acts as a buffer to fine tune absorption on a day-to-day basis. For the buffering capacity, the ability to alter DMT1 levels rapidly is particularly important. The major splice variant of the DMT1 mRNA in the gut contains an iron responsive element (IRE) in its 3' UTR and the binding of the iron-dependent iron regulatory proteins 1 and 2 (IRP1/2) has been considered to be important in stabilizing the DMT1 message. Recently, Galy et al.  have provided experimental proof that this is the case by generating intestine-specific IRP1 and IRP2 double knockouts in mice. The resulting animals have a strong reduction in DMT1 expression and have reduced iron intake. A surprising finding from these studies is that FPN protein was upregulated in the knockout mice. FPN mRNA contains an IRE in its 5' UTR, which mediates translational control in some tissues, but this was thought to be inactive in the gut, so this observation requires further investigation.
Another exciting discovery applicable to enterocytes is the identification of a potential iron chaperone that delivers iron to ferritin [5••]. Chaperones for transporting copper intracellularly are well known and it has often been postulated that similar proteins might exist for iron, but the recent study by Shi et al. [5••] is the first evidence that this is the case. They showed that poly (rC) binding protein 1 (PCBP1) was able to facilitate iron loading onto human ferritin expressed in the yeast Saccharomyces cerevisiae and subsequently found that it could perform a similar function in mammalian cells. As PCBP1 appears to be ubiquitously expressed in mammalian cells, it is likely to play a similar role in intestinal enterocytes.
Systemic regulation of iron absorption
The resurgence in interest in iron absorption in recent years has been driven by the identification of the liver-derived peptide hepcidin as a central regulator of body iron homeostasis . Hepcidin expression is inversely related to body iron demand, being increased in times of iron sufficiency and decreased when iron requirements are high. Hepcidin in turn acts on its downstream target cells to regulate how much iron they release into the plasma. Important cellular targets include intestinal enterocytes, macrophages and hepatocytes. Hepcidin limits cellular iron efflux by binding to the iron exporter FPN and facilitating its internalization and degradation. The mechanism by which hepcidin acts on FPN has recently been investigated in detail and it sequentially involves tyrosine phosphorylation on one of the FPN cytoplasmic domains, internalization of the protein, dephosphorylation, ubiquitination and trafficking the late endosome/lysosome compartment for degradation [7•].
As the principal target of hepcidin action has been identified, there has been increasing focus on how hepcidin itself is regulated. It is not surprising that the same stimuli that have been known for many years to regulate intestinal iron absorption –alterations in body iron stores, changes in the rate of erythropoiesis, hypoxia and inflammation – are the same factors that regulate HAMP, the gene encoding hepcidin.
The role played by the analysis of inherited disorders of iron metabolism in helping us to understand iron transport and its regulation cannot be underestimated, and some of the key defects investigated are summarized in Table 1. Mutations in the gene encoding hepcidin (HAMP) effectively mimic an iron deficiency situation and lead to an increase in iron absorption and a severe early onset iron overload disease known as juvenile hemochromatosis. Mutations in several other genes, including those encoding hemojuvelin (HJV), HFE and transferrin receptor 2 (TfR2) also lead to iron loading syndromes that are phenotypically very similar to that associated with hepcidin deficiency (although the disease severity may vary), and these disorders do indeed have low or absent hepcidin expression . Thus, hepcidin, HJV, HFE and TfR2 are all parts of the same regulatory pathway, with HJV, HFE and TfR2 being upstream regulators of hepcidin. This regulatory system is based in the liver and specifically in the hepatocytes as this is the major site of expression of most of the regulatory proteins. Indeed, the recent confirmation that HFE exerts its effects in hepatocytes [9•,10••] has resolved a longstanding issue in this field.
The upstream regulators respond to external signals and alter hepcidin expression through one or more signal transduction pathways, but perhaps the most profound insights have come from the analysis of HJV function. HJV is a member of the repulsive guidance molecule family of proteins that signal through the multifunctional bone morphogenetic protein (BMP)-SMAD signaling pathway to modulate cellular activity. HJV acts as a cofactor for activation of the BMP receptors by several BMPs (notably BMPs 2, 4 and 9) [11•,12••] (Fig. 2). Activated BMP receptors phosphorylate SMAD1/5/8, which in turn dimerize with the DNA binding protein SMAD4. The complex subsequently translocates to the nucleus where it influences the expression of target genes such as the gene encoding hepcidin. Independently, it was shown that Smad4-knockout mice developed significant iron loading and had reduced hepcidin expression , confirming the importance of this pathway in regulating body iron homeostasis.
The role of HJV in the activation of the BMP-SMAD pathway is far from resolved. Membrane-bound HJV acts to stimulate signaling through the BMP-SMAD pathway, whereas a soluble form of HJV (sHJV) acts to inhibit signaling [11•]. The source of sHJV is unclear, though skeletal muscle is certainly a strong candidate. HJV is most strongly expressed in this tissue and skeletal muscle contains one of the largest pools of iron in the body. sHJV is released from the mature HJV protein through the action of the protease furin, and recent data have shown that furin activity can be increased by iron deficiency and hypoxia [14•], both conditions that increase iron absorption. Increased furin activity would increase the level of circulating sHJV, which in turn would inhibit hepcidin activation and facilitate increased iron flux into the plasma. The inhibition of sHJV release in response to increased iron also appears to require the protein neogenin .
The severity of the phenotype that results from mutations in HJV suggests that the BMP-SMAD signaling pathway plays a central role in the regulation of hepcidin, but whether HFE and TfR2 also regulate hepcidin through this pathway is unknown. HFE and TfR2 proteins appear to be involved in sensing the body's iron requirements and they likely respond to levels of diferric transferrin in the plasma . HFE binds to TfR1, the main plasma membrane transferrin binding protein, but can be displaced when the level of circulating diferric transferrin increases, such as under iron loading conditions. This ‘free’ HFE is then able to induce hepcidin expression, perhaps through its interaction with TfR2. Schmidt et al.[17•] have recently described a series of elegant experiments using transgenic and knockin mouse models to confirm that HFE is able to alter hepcidin expression when not bound to TfR1.
Hepcidin is also able to respond to several other stimuli (Fig. 2). Proinflammatory cytokines such as IL-6 can induce expression of the HAMP gene through the janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway . Interestingly, the response of HAMP to an inflammatory stimulus is abrogated in Smad4-knockout mice, indicating that an intact SMAD pathway is required for this response. Consistent with this, it has recently been shown that although the BMP-SMAD and JAK-STAT pathways recognize separate cis-acting elements in the HAMP promoter, the SMAD binding element is necessary for an appropriate response to IL-6 [19•,20••]. Hepcidin expression can also be reduced by hypoxia and this response has recently been shown to be mediated by the von Hippel-Lindau/hypoxia-inducible transcription factor (VHL/HIF) pathway [21•,22].
New players in hepcidin regulation
As the main sink for iron in the body is the erythroid marrow where the iron is used for hemoglobin synthesis, it is not surprising that elevated erythropoiesis leads to a reduction in hepcidin expression and a concomitant increase in iron absorption. This response in part reflects lower diferric transferrin levels and hypoxia, but erythroid-specific factors may also be involved. One candidate for such a factor is growth differentiation factor 15 (GDF15). GDF15 is produced during erythroid cell production and it has been shown to negatively regulate hepcidin expression in vitro[23•]. In addition, GDF15 levels are raised under conditions such as thalassemia in which iron absorption is increased. Despite these encouraging observations, a recent study [24•], in which patients undergoing hematopoietic stem cell transplantation were followed up prospectively, failed to find a correlation between GDF15 levels and hepcidin, though the latter correlated well with other markers of erythropoiesis. Thus, the contribution of GDF15 requires further investigation.
The most recent addition to the suite of proteins involved in hepcidin regulation is matriptase-2 (encoded by the TMPRSS6 gene). A role for this membrane-bound serine protease in iron homeostasis was identified independently by two groups. One identified it as the gene mutated in the mask mouse mutant, a strain of mice with an inherited hypochromic, microcytic anemia [25••], whereas the other group identified it as the gene affected in cases of refractory iron deficiency anemia in humans [26••]. Since then a number of other groups have identified TMPRSS6 mutations in iron-deficient patients [27,28] and targeted disruption of the Tmprss6 gene in mice has been reported [29•]. In both mice and humans, hepcidin levels were inappropriately high when TMPRSS6 was mutated, suggesting that matripase-2 acts as a repressor of hepcidin expression under normal conditions. Consistent with this, one of the groups demonstrated that overexpression of TMPRSS6 suppresses HAMP promoter activity and that the protease domain is required for a full effect [25••]. Matriptase-2 could be involved in the upstream proteolytic processing of one of the previously known regulators of hepcidin (e.g. BMP-SMAD pathway, HIF1-α, GDF15), though Du et al.[25••] suggest that it may mediate a novel, yet clearly very important, pathway.
What is new for the clinician?
The identification of hepcidin as the central regulator of iron homeostasis immediately raised the possibility of diagnostic and therapeutic applications. As the hepcidin concentration reflects body iron requirements, the ability to measure plasma or urinary hepcidin could potentially become the single most important test for monitoring iron status. However, progress in developing a simple, inexpensive and widely available hepcidin assay has been slow. This largely reflects the difficulties experienced in raising antibodies against the mature hepcidin peptide. Prohepcidin is more immunogenic, but its measurement has not proved a particularly effective surrogate for the mature peptide. Mass spectrometry approaches to measuring hepcidin have been developed in several centers and these are reasonably effective [30,31], but it is not a method that is currently widely available. Despite the difficulties in raising hepcidin antibodies, some suitable reagents are available and have been used to quantitate hepcidin in a research setting . Hopefully, soon we will have a commercial immunoassay available for the peptide.
Hepcidin also has considerable potential as a therapeutic agent. Administering hepcidin to patients with inherited forms of iron overload could replace the need for regular phlebotomy and this has been proved in principle in animal studies. Alternatively, blocking hepcidin synthesis or action could have application in preventing the anemia of chronic disease. Potential targets include the BMP-SMAD pathway and matriptase-2. Nevertheless, this is another area of hepcidin biology where we have not seen rapid progress and no hepcidin clinical trials have been published to date.
Our understanding of the regulation of intestinal iron absorption has progressed rapidly in recent years with the definition of the factors that regulate the iron homeostasis hormone hepcidin and their mechanisms of action. Over the next few years, we can look forward to these exciting basic discoveries being translated into improved diagnostic and therapeutic modalities for patients with disturbances in iron metabolism that lead to altered iron absorption. What has not progressed as rapidly is our understanding of the molecular details of iron transport across the enterocyte and this is certainly a fruitful area for future investigations.
G.J.A. is supported by a Senior Research Fellowship from the National Health and Medical Research Council of Australia and D.M.F. is supported by a Bushell Postdoctoral Fellowship from the Gastroenterological Society of Australia.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 169–170).
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