Minihepcidins prevent iron overload in a hepcidin-deficient mouse model of severe hemochromatosis.

The deficiency of hepcidin, the hormone that controls iron absorption and its tissue distribution, is the cause of iron overload in nearly all forms of hereditary hemochromatosis and in untransfused iron-loading anemias. In a recent study, we reported the development of minihepcidins, small drug-like hepcidin agonists. Here we explore the feasibility of using minihepcidins for the prevention and treatment of iron overload in hepcidin-deficient mice. An optimized minihepcidin (PR65) was developed that had superior potency and duration of action compared with natural hepcidin or other minihepcidins, and favorable cost of synthesis. PR65 was administered by subcutaneous injection daily for 2 weeks to iron-depleted or iron-loaded hepcidin knockout mice. PR65 administration to iron-depleted mice prevented liver iron loading, decreased heart iron levels, and caused the expected iron retention in the spleen and duodenum. At high doses, PR65 treatment also caused anemia because of profound iron restriction. PR65 administration to hepcidin knockout mice with pre-existing iron overload had a more moderate effect and caused partial redistribution of iron from the liver to the spleen. Our study demonstrates that minihepcidins could be beneficial in iron overload disorders either used alone for prevention or possibly as adjunctive therapy with phlebotomy or chelation.

Inhibition of hepcidin transcription by growth factors.

UNLABELLED: The hepatic peptide hormone hepcidin controls the duodenal absorption of iron, its storage, and its systemic distribution. Hepcidin production is often insufficient in chronic hepatitis C and alcoholic liver disease, leading to hyperabsorption of iron and its accumulation in the liver. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mediate hepatic regeneration after liver injury. We examined the effect of these growth factors on hepcidin synthesis by hepatocytes. HGF and EGF treatment of primary mouse hepatocytes, as well as EGF administration in mice, suppressed hepcidin messenger RNA (mRNA) synthesis. The suppression of hepcidin by these growth factors was transcriptional, and was mediated by a direct effect of HGF and EGF on the bone morphogenetic protein (BMP) pathway regulating hepcidin synthesis. We further show that growth factors interfered with nuclear localization of activated sons of mothers against decapentaplegic (Smad) and increased the nuclear pool of the BMP transcriptional corepressor TG-interacting factor (TGIF). In a kinase screen with small-molecule kinase inhibitors, inhibitors in the PI3 kinase pathway and in the mitogen-activated ERK kinase/extracellular signal-regulated kinase (MEK/ERK) pathway prevented HGF suppression of hepcidin in primary mouse hepatocytes. CONCLUSION: HGF and EGF suppress hepatic hepcidin synthesis, in part through PI3 kinase MEK/ERK kinase pathways which may be modulating the nuclear localization of BMP pathway transcriptional regulators including activated Smads1/5/8 and the corepressor TGIF. EGF, HGF, and possibly other growth factors that activate similar pathways may contribute to hepcidin suppression in chronic liver diseases, promote iron accumulation in the liver, and exacerbate the destructive disease processes.

Reduced serum hepcidin levels in patients with chronic hepatitis C.

BACKGROUND/AIMS: Patients with chronic hepatitis C (CHC) often have increased liver iron, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is the major regulator of iron metabolism and inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (s-hepcidin). METHODS: Using a recently validated immunoassay, we measured s-hepcidin levels in 81 untreated CHC patients and 57 controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score. RESULTS: s-hepcidin was significantly lower in CHC patients than in controls (geometric means with 95% confidence intervals: 33.7, 21.5-52.9 versus 90.9, 76.1-108.4 ng/mL, respectively; p<0.001). In CHC patients, s-hepcidin significantly correlated with serum ferritin and histological total iron score, but not with s-interleukin-6. After stratification for ferritin quartiles, s-hepcidin increased significantly across quartiles in both controls and CHC patients (chi for trend, p<0.001). However, in CHC patients, s-hepcidin was significantly lower than in controls for each corresponding quartile (analysis of variance, p<0.001). CONCLUSIONS: These results, together with very recent studies in animal and cellular models, indicate that although hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by hepatitis C virus is likely an important factor in liver iron accumulation in this condition.

Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.

In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.

Soluble hemojuvelin is released by proprotein convertase-mediated cleavage at a conserved polybasic RNRR site.

As the principal iron-regulatory hormone, hepcidin plays an important role in systemic iron homeostasis. The regulation of hepcidin expression by iron loading appears to be unexpectedly complex and has attracted much interest. The GPI-linked membrane protein hemojuvelin (GPI-hemojuvelin) is an essential upstream regulator of hepcidin expression. A soluble form of hemojuvelin (s-hemojuvelin) exists in blood and acts as antagonist of GPI-hemojuvelin to downregulate hepcidin expression. The release of s-hemojuvelin is negatively regulated by both transferrin-bound iron (holo-Tf) and non-transferrin-bound iron (FAC), indicating s-hemojuvelin could be one of the mediators of hepcidin regulation by iron. In this report, we investigate the proteinase involved in the release of s-hemojuvelin and show that s-hemojuvelin is released by a proprotein convertase through the cleavage at a conserved polybasic RNRR site.

Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and BMP2/4.

The peptide hormone hepcidin is the principal regulator of systemic iron homeostasis. We examined the pathway by which iron stimulates the production of hepcidin. In humans who ingested 65 mg of iron, the increase in transferrin saturation preceded by hours the increase in urinary hepcidin excretion. Increases in urinary hepcidin concentrations were proportional to the increment in transferrin saturation. Paradoxically, in previous studies in primary hepatocytes and cell lines, hepcidin response to iron or iron transferrin was not observed. We now report that freshly isolated murine primary hepatocytes responded to holotransferrin but not apotransferrin by increasing hepcidin mRNA. Hepcidin increase was not due to contamination of the transferrin preparations by endotoxin, a potent pathologic stimulus of hepcidin synthesis. Using this culture system, we showed that holotransferrin concentrations regulate hepcidin mRNA concentrations through a hemojuvelin/BMP2/4-dependent pathway. Although BMP9 is known to be expressed in the liver and potently increased the basal concentrations of hepcidin mRNA, it did not interact with hemojuvelin, and interference with its signaling pathway did not affect iron regulation. Fresh primary hepatocytes constitute a sufficient system for the regulation of hepcidin by physiologic iron stimuli and will greatly facilitate studies of major disorders of iron homeostasis.

Differential requirement for TANK-binding kinase-1 in type I interferon responses to toll-like receptor activation and viral infection.

TANK-binding kinase-1 (TBK1) and the inducible IkappaB kinase (IKK-i) have been shown recently to activate interferon (IFN) regulatory factor-3 (IRF3), the primary transcription factor regulating induction of type I IFNs. Here, we have compared the role and specificity of TBK1 in the type I IFN response to lipopolysaccharide (LPS), polyI:C, and viral challenge by examining IRF3 nuclear translocation, signal transducer and activator of transcription 1 phosphorylation, and induction of IFN-regulated genes. The LPS and polyI:C-induced IFN responses were abolished and delayed, respectively, in macrophages from mice with a targeted disruption of the TBK1 gene. When challenged with Sendai virus, the IFN response was normal in TBK1(-/-) macrophages, but defective in TBK1(-/-) embryonic fibroblasts. Although both TBK1 and IKK-i are expressed in macrophages, only TBK1 but not IKK-i was detected in embryonic fibroblasts by Northern blotting analysis. Furthermore, the IFN response in TBK1(-/-) embryonic fibroblasts can be restored by reconstitution with wild-type IKK-i but not a mutant IKK-i lacking kinase activity. Thus, our studies suggest that TBK1 plays an important role in the Toll-like receptor-mediated IFN response and is redundant with IKK-i in the response of certain cell types to viral infection.