Carole Beaumont

The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation

The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Severe iron deficiency anemia in transgenic mice expressing liver hepcidin

We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.

Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type.

When adult rat hepatocytes were co-cultured with another liver epithelial cell type in a medium supplemented or not with fetal calf serum (FCS), it was found that 1. They survived for more than 2 months 2. Albumin secretion levels remained high over the whole culture period 3. Decreased secretion might be reversed 4. This protein secretion activity appeared to be dependent upon both the presence of cell-cell contacts and the production of an extracellular material. The results demonstrate for the first time long-term stabilization and reversibility of a specific function (albumin secretion) at high levels by adult hepatocytes cultured in serum-free medium and suggest that both the presence of other liver cell type(s) and the production of an extracellular matrix are needed for the maintenance of specific functions in cultured hepatocytes.

Ferritin Heavy Chain Upregulation by NF-κB Inhibits TNFα-Induced Apoptosis by Suppressing Reactive Oxygen Species

Abstract During inflammation, NF-κB transcription factors antagonize apoptosis induced by tumor necrosis factor (TNF)α. This antiapoptotic activity of NF-κB involves suppressing the accumulation of reactive oxygen species (ROS) and controlling the activation of the c-Jun N-terminal kinase (JNK) cascade. However, the mechanism(s) by which NF-κB inhibits ROS accumulation is unclear. We identify ferritin heavy chain (FHC)—the primary iron storage factor—as an essential mediator of the antioxidant and protective activities of NF-κB. FHC is induced downstream of NF-κB and is required to prevent sustained JNK activation and, thereby, apoptosis triggered by TNFα. FHC-mediated inhibition of JNK signaling depends on suppressing ROS accumulation and is achieved through iron sequestration. These findings establish a basis for the NF-κB-mediated control of ROS induction and identify a mechanism by which NF-κB suppresses proapoptotic JNK signaling. Our results suggest modulation of FHC or, more broadly, of iron metabolism as a potential approach for anti-inflammatory therapy.

Constitutive hepcidin expression prevents iron overload in a mouse model of hemochromatosis.

Hereditary hemochromatosis is a prevalent genetic disorder of iron hyperabsorption leading to hyperferremia, tissue iron deposition and complications including cirrhosis, hepatocarcinoma, cardiomyopathy and diabetes. Most individuals affected with hereditary hemochromatosis are homozygous with respect to a missense mutation that disrupts the conformation of HFE, an atypical HLA class I molecule (ref. 1; OMIM 235200). Mice lacking Hfe or producing a C282Y mutant Hfe protein develop hyperferremia and have high hepatic iron levels. In both humans and mice, hereditary hemochromatosis is associated with a paucity of iron in reticuloendothelial cells. It has been suggested that HFE modulates uptake of transferrin-bound iron by undifferentiated intestinal crypt cells, thereby programming the absorptive capacity of enterocytes derived from these cells; however, this model is unproven and controversial. Hepcidin, a peptide hormone (HAMP; OMIM 606464), seems to act in the same regulatory pathway as HFE. Although expression of mouse Hamp is normally greater during iron overload, Hfe−/− mice have inappropriately low expression of Hamp. We crossed Hfe−/− mice with transgenic mice overexpressing Hamp and found that Hamp inhibited the iron accumulation normally observed in the Hfe−/− mice. This argues against the crypt programming model and suggests that failure of Hamp induction contributes to the pathogenesis of hemochromatosis, providing a rationale for the use of HAMP in the treatment of this disease.

Neuromelanin associated redox‐active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinsons disease is most extensive in the subpopulation of melanized‐neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post‐mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox‐active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)‐aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (− 70%) in the number of melanized‐neurones and an increased non‐heme (Fe3+) iron content as compared with a group of matched‐control subjects. The level of redox activity detected in neuromelanin‐aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized‐neurones. A possible consequence of an overloading of neuromelanin with redox‐active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinsons disease.

C-Terminal Deletions in the ALAS2 Gene Lead to Gain of Function and Cause X-linked Dominant Protoporphyria without Anemia or Iron Overload

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

Intestinal DMT1 Cotransporter Is Down-regulated by Hepcidin via Proteasome Internalization and Degradation

BACKGROUNDS & AIMS The mechanism by which hepcidin regulates iron export from macrophages has been well established and is believed to involve degradation of ferroportin. However, in the small intestine, hepcidins mechanisms of action are not known. We studied human polarized intestinal (Caco-2/TC7) cells and mouse duodenal segments, ex vivo, to investigate the molecular mechanisms by which hepcidin down-regulates intestinal transepithelial iron transport. METHODS Iron transport was analyzed using ⁵⁵FeNTA. Expression of Divalent Metal Transporter 1 (DMT1) and ferroportin was evaluated by reverse-transcription quantitative polymerase chain reaction and immunoblotting. Videomicroscopy analysis was performed on live cells that expressed either DMT1 or ferroportin fused to green fluorescent protein. RESULTS In Caco-2/TC7 cells, physiologic doses of hepcidin (50-1000 nmol/L) inhibited transport of ⁵⁵Fe in a dose-dependent manner; a half-maximum effect was observed at 75-100 nmol/L. However, 200 nmol/L hepcidin induced a significant decrease in DMT1 protein expression but no change in ferroportin protein levels, unlike macrophages. This result was confirmed ex vivo in isolated duodenal segments: 200 nmol/L hepcidin induced a significant reduction in iron transport and DMT1 protein levels but no change in ferroportin levels. In Caco-2/TC7 cells, the effect of hepcidin on the DMT1 protein level was completely abolished in the presence of a proteasome inhibitor (MG-132); DMT1 ubiquitination was induced by the addition of hepcidin. CONCLUSIONS An acute increase in hepcidin concentration reduces intestinal iron absorption through ubiquitin-dependent proteasome degradation of DMT1.

Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia

Differentiating agents have been proposed to overcome the impaired cellular differentiation in acute myeloid leukemia (AML). However, only the combinations of all-trans retinoic acid or arsenic trioxide with chemotherapy have been successful, and only in treating acute promyelocytic leukemia (also called AML3). We show that iron homeostasis is an effective target in the treatment of AML. Iron chelating therapy induces the differentiation of leukemia blasts and normal bone marrow precursors into monocytes/macrophages in a manner involving modulation of reactive oxygen species expression and the activation of mitogen-activated protein kinases (MAPKs). 30% of the genes most strongly induced by iron deprivation are also targeted by vitamin D3 (VD), a well known differentiating agent. Iron chelating agents induce expression and phosphorylation of the VD receptor (VDR), and iron deprivation and VD act synergistically. VD magnifies activation of MAPK JNK and the induction of VDR target genes. When used to treat one AML patient refractory to chemotherapy, the combination of iron-chelating agents and VD resulted in reversal of pancytopenia and in blast differentiation. We propose that iron availability modulates myeloid cell commitment and that targeting this cellular differentiation pathway together with conventional differentiating agents provides new therapeutic modalities for AML.