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Iron overload as a result of blood transfusions and excessive intestinal iron absorption can be a complication of chronic anemias such as β-thalassemia. Inappropriately low levels of hepcidin, a negative regulator of iron absorption and recycling, underlie the pathophysiology of the intestinal hyperabsorption. In this issue of the JCI, Gardenghi et al. demonstrate that increasing hepcidin expression to induce iron deficiency in murine β-thalassemia not only mitigates the iron overload, but also the severity of the anemia. These data illustrate the therapeutic potential of modulating hepcidin expression in diseases associated with altered iron metabolism.
Erythropoiesis consumes the majority of the iron present in the human body (1). Most of this iron is obtained from the recycling of effete red blood cells by macrophages found in the liver, spleen, and bone marrow. Interruption of iron export from macrophages leads to functional iron deficiency and iron-limited erythropoiesis. At equilibrium, only a small amount of iron is absorbed in the duodenum from the diet each day. Further, there is no physiologically regulated mechanism of eliminating excess iron from the body. Consequently, the proper regulation of dietary iron absorption as well as iron recycling is essential to maintaining iron homeostasis and to sustaining erythropoiesis.
The peptide hormone hepcidin is the key regulator of iron absorption and recycling (2). Secreted predominantly by hepatocytes, hepcidin binds to and induces the internalization and degradation of ferroportin, an iron export protein present on the cell surface of macrophages and duodenal enterocytes. In this manner, hepcidin limits iron recycling and absorption, thereby controlling body iron stores and the availability of iron for erythropoiesis. As a negative regulator, hepcidin expression is inversely correlated with body iron stores. Importantly, hepcidin expression is inhibited by erythropoietic activity through an as-yet-unknown mechanism conceptualized as the “erythroid regulator” of iron homeostasis. This signal is particularly strong in individuals with anemia, such as those with β-thalassemia.
Hemoglobin is composed of two α-globin and two β-globin chains, with each chain having one associated heme moiety. β-thalassemia is an inherited disorder of β-globin synthesis due to mutations that result in decreased levels of a structurally normal β-globin protein. In β-thalassemia, excess unpaired α-globin chains resulting from the β-globin synthesis defect are toxic to erythroid precursors, causing their premature death in the bone marrow. Such abortive erythropoiesis is termed “ineffective erythropoiesis” and is a particularly potent inducer of the erythroid regulator of iron homeostasis. Chronically low levels of hepcidin lead to excessive dietary iron absorption and iron overload, which manifests clinically as liver disease, cardiomyopathy, and endocrinopathies. The hepcidin suppression caused by ineffective erythropoiesis is so strong that individuals with β-thalassemia intermedia, a moderately severe form of β-thalassemia in which patients do not require frequent blood transfusions to survive, can develop clinically significant iron overload. In severe β-thalassemia (β-thalassemia major) chronic blood transfusions, employed to correct the anemia and suppress ineffective erythropoiesis, lead to massive iron overload associated with high hepcidin levels. In either case, iron overload is a major cause of morbidity and mortality in these patients, necessitating pharmacological iron chelation.
Therein lies the paradox of iron homeostasis and hepcidin in the setting of ineffective erythropoiesis and, in particular, β-thalassemia intermedia: iron is required to make red blood cells to correct the anemia, and the body responds to ineffective erythropoiesis by decreasing hepcidin levels to provide iron, yet the response is in some manner maladaptive, as it leads to chronic iron overload and its attendant toxicities. If hepcidin levels are inappropriately low for the degree of iron stores in β-thalassemia intermedia, then increasing them might attenuate the severity of iron overload. In this issue of the JCI, Gardenghi et al. test just that (3).
Hypothesizing that β-thalassemic mice absorb more dietary iron than is needed for erythropoiesis, Gardenghi et al. placed β-thalassemic mice on a low-iron diet (3). As expected, iron levels significantly decreased. Remarkably, and somewhat unexpectedly, however, erythropoiesis was relatively unaffected and the anemia and splenomegaly actually improved. Next, the authors reasoned that increasing hepcidin expression could have the same effect as limiting dietary iron. To this end, they took several approaches. First, they used viral transduction to overexpress the hemochromatosis gene Hfe, which encodes a membrane protein that promotes hepcidin expression, in the livers of β-thalassemic mice and demonstrated a decrease in tissue iron content without any adverse effects on erythropoiesis. Second, they bred the anemic animals to a transgenic line that constitutively expresses low levels of hepcidin. β-thalassemic mice carrying the transgene demonstrated an improvement in their anemia and decreased tissue iron content at one and five months of age.
A major determinant of the pathophysiology of β-thalassemia is the accumulation of precipitated unpaired α-globin chains and membrane damage caused by partially denatured, oxidized hemoglobin derivatives such as hemichrome. In the hepcidin-expressing transgenic mice, Gardenghi et al. found lower levels of membrane-associated α-globin chains as well as decreased red blood cell reactive oxygen species (3), compatible with the notion that slightly increased hepcidin levels ultimately relieved the proximal cause of cell damage and death. Whether the observed improvement in the anemia was due to less ineffective erythropoiesis, increased red blood cell lifespan, or a combination thereof, remains to be determined. However, it seems likely that the effect may be a consequence of a reduced rate of α- compared with β-globin synthesis induced by iron deficiency (4). Intriguingly, several mice expressing very high levels of hepcidin had more severe anemia, indicating that there is very likely a narrow therapeutic window of hepcidin efficacy in this disease model.
The paper by Gardenghi et al. (3) is timely in that it complements a recent publication by Li et al. (5). In that paper, the authors demonstrated that serial injections of transferrin, the iron-binding protein in the serum that is essential for iron delivery to erythroid progenitors, leads to increased hepcidin expression and significant improvements in anemia in the same mouse model of β-thalassemia. While Li et al. explained their findings by concluding that transferrin is a limiting factor in β-thalassemia (5), the work of Gardenghi et al. suggests that downstream effects on hepcidin or restricted erythroid iron delivery could be the major contributing factor (3).
Altering hepcidin levels and/or activity has also proven beneficial in disorders other than β-thalassemia (Figure (Figure1).1). Transgenic overexpression of hepcidin and injections of synthetic hepcidin both lead to reduced tissue iron levels in mouse models of genetic hemochromatosis, a group of diseases of hepcidin deficiency and iron overload generally due to mutations in factors required for hepcidin expression (6, 7). Modulation of hepcidin activity and/or levels may also be of clinical utility in inflammatory disorders associated with anemia (i.e., the anemia of inflammation), in which elevated levels of proinflammatory cytokines such as IL-6 lead to hepcidin overexpression and promote iron-restricted erythropoiesis. Indeed, administration of hepcidin-specific antibodies increases serum iron levels in a mouse model of the anemia of inflammation (8). In addition, administration of IL-6–specific antibodies reduces hepcidin levels and improves anemia in a monkey collagen-induced arthritis model (9) as well as reduces serum hepcidin levels in human patients with Castleman disease, a lymphoproliferative disorder associated with high levels of IL-6 (10). Targeting downstream mediators of inflammation-induced hepcidin expression such as the JAK/STAT signaling pathway may also prove efficacious in the anemia of inflammation. While STAT3 inhibitors inhibit hepcidin expression in vitro, the cytokine oncostatin M, the hormone leptin, and the cytokine leukemia inhibitory factor (LIF) all activate the STAT pathway in vitro; oncostatin M has also been shown to increase hepcidin expression in mice (11–14).
Hepcidin levels can also be altered by targeting a bone morphogenetic protein–dependent (BMP-dependent) pathway known to be central to regulation of hepcidin expression. This pathway involves a multitude of extracellular, membrane-bound, and intracellular factors (2). Growth differentiation factor 15 (Gdf15) and twisted gastrulation homolog 1 (Twsg1), both BMP antagonists produced by erythroblasts and putative components of the erythroid regulator, inhibit hepcidin expression in vitro, although their physiologic relevance has not been established definitively (15). Administration of dorsomorphin, a BMP type I receptor inhibitor, decreases hepcidin expression in mice (16). While dorsomorphin and its more receptor-selective derivative compound LDN-193189 have not been tested in humans, these agents represent an exciting potential therapy for a variety of disorders associated with inappropriately high hepcidin levels, such as the anemia of inflammation. Likewise, the hepatocyte BMP receptor complex and signaling cascade might equally be therapeutic targets in conditions associated with hepcidin excess. For example, hemojuvelin (HJV; also known as HFE2), which is mutated in a severe form of juvenile onset hemochromatosis, is required for hepcidin expression and believed to act as a BMP coreceptor (2). Its levels are regulated by the activity of two other proteins, transmembrane protease serine 6 (Tmprss6) and neogenin, which are themselves potential drug targets (17). Mutations in Tmprss6 cause iron-responsive iron deficiency anemia (IRIDA), a form of inherited microcytic anemia due to hepcidin overexpression, and lead to excessive hepcidin production in humans and mice, while neogenin-deficient mice exhibit hepcidin deficiency and iron overload. Tmprss6 cleaves HJV from the cell surface, thereby liberating a soluble form of HJV, while neogenin inhibits secretion of HJV from hepatocytes. Soluble HJV inhibits hepcidin expression in vitro, but this agent has yet to be tested in vivo (18). Finally, liver-specific deletion of the downstream transcription factor SMAD4 leads to hepcidin deficiency and iron overload in mice, suggesting that antagonism of SMAD4 activity could downregulate hepcidin expression (19).
Overall, the work of Gardenghi et al. (3) is exciting in that it represents a proof of concept that diseases associated with iron overload can be treated by modulating hepcidin expression and/or activity. Furthermore, their data suggest that manipulating this pathway may be a method of not only treating the secondary iron overload associated with β-thalassemia intermedia, but also of ameliorating the anemia itself. Because of its short half-life, pharmacologic means of increasing the expression of endogenous hepcidin and/or its downstream activity, rather than administering hepcidin itself, would be the ideal therapeutic. Furthermore, in practical terms, a multipronged approach involving hepcidin modulators and iron chelation might be the most effective strategy. Whatever the eventual method might be, it is clear that hepcidin is now a well-validated target earnestly awaiting human application.
The authors thank David G. Nathan and H. Franklin Bunn for helpful discussions. M.D. Fleming is supported by NIH R01 DK080011. T.B. Bartnikas is supported by NIH K99 DK084122.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2010;120(12):4187–4190. doi:10.1172/JCI45043.
See the related article beginning on page 4466.