Engineering Peptide Inhibitors of the HFE-Transferrin Receptor 1 Complex.

The protein HFE (homeostatic iron regulator) is a key regulator of iron metabolism, and mutations in HFE underlie the most frequent form of hereditary haemochromatosis (HH-type I). Studies have shown that HFE interacts with transferrin receptor 1 (TFR1), a homodimeric type II transmembrane glycoprotein that is responsible for the cellular uptake of iron via iron-loaded transferrin (holo-transferrin) binding. It has been hypothesised that the HFE/TFR1 interaction serves as a sensor to the level of iron-loaded transferrin in circulation by means of a competition mechanism between HFE and iron-loaded transferrin association with TFR1. To investigate this, a series of peptides based on the helical binding interface between HFE and TFR1 were generated and shown to significantly interfere with the HFE/TFR1 interaction in an in vitro proximity ligation assay. The helical conformation of one of these peptides, corresponding to the α1 and α2 helices of HFE, was stabilised by the introduction of sidechain lactam "staples", but this did not result in an increase in the ability of the peptide to disrupt the HFE/TFR1 interaction. These peptides inhibitors of the protein-protein interaction between HFE and TFR1 are potentially useful tools for the analysis of the functional role of HFE in the regulation of hepcidin expression.

Genetic Diagnosis in Hereditary Hemochromatosis: Discovering and Understanding the Biological Relevance of Variants.

BACKGROUND: Hereditary hemochromatosis (HH) is a genetic disease, leading to iron accumulation and possible organ damage. Patients are usually homozygous for p. Cys282Tyr in the homeostatic iron regulator gene but may have mutations in other genes involved in the regulation of iron. Next-generation sequencing is increasingly being utilized for the diagnosis of patients, leading to the discovery of novel genetic variants. The clinical significance of these variants is often unknown. CONTENT: Determining the pathogenicity of such variants of unknown significance is important for diagnostics and genetic counseling. Predictions can be made using in silico computational tools and population data, but additional evidence is required for a conclusive pathogenicity classification. Genetic disease models, such as in vitro models using cellular overexpression, induced pluripotent stem cells or organoids, and in vivo models using mice or zebrafish all have their own challenges and opportunities when used to model HH and other iron disorders. Recent developments in gene-editing technologies are transforming the field of genetic disease modeling. SUMMARY: In summary, this review addresses methods and developments regarding the discovery and classification of genetic variants, from in silico tools to in vitro and in vivo models, and presents them in the context of HH. It also explores recent gene-editing developments and how they can be applied to the discussed models of genetic disease.

In vitro identification and characterisation of iron chelating catechol-containing natural products and derivatives.

Iron is an essential component for multiple biological processes. Its regulation within the body is thus tightly controlled. Dysregulation of iron levels within the body can result in several disorders associated with either excess iron accumulation, including haemochromatosis and thalassaemia, or iron deficiency. In cases of excess body iron, therapy involves depleting body iron levels either by venesection, typically for haemochromatosis, or using iron chelators for thalassemia. However, the current chelation options for people with iron overload are limited, with only three iron chelators approved for clinical use. This presents an opportunity for improved therapeutics to be identified and developed. The aim of this study was to examine multiple compounds from within the Davis open access natural product-based library (512 compounds) for their ability to chelate iron. In silico analysis of this library initially identified nine catechol-containing compounds and two closely related compounds. These compounds were subsequently screened using an in vitro DNA breakage assay and their ability to chelate biological iron was also examined in an iron-loaded hepatocyte cellular assay. Toxicity was assessed in hepatocyte and breast cancer cell lines. One compound, RAD362 [N-(3-aminopropyl)-3,4-dihydroxybenzamide] was able to protect against DNA damage, likely through the prevention of free radicals generated via the Fenton reaction; RAD362 treatment resulted in decreased ferritin protein levels in iron-loaded hepatocytes. Lastly, RAD362 resulted in significantly less cell death than the commonly used iron chelator deferoxamine. This is the first study to identify compound RAD362 as an iron chelator and potential therapeutic.

Increased frequency of GNPAT p.D519G in compound HFE p.C282Y/p.H63D heterozygotes with elevated serum ferritin levels.

Glyceronephosphate O-acyltransferase (GNPAT) p.D519G (rs11558492) was identified as a genetic modifier correlated with more severe iron overload in hemochromatosis through whole-exome sequencing of HFE p.C282Y homozygotes with extreme iron phenotypes. We studied the prevalence of p.D519G in HFE p.C282Y/p.H63D compound heterozygotes, a genotype associated with iron overload in some patients. Cases were Australian participants with elevated serum ferritin (SF) levels ≥300µg/L (males) and ≥200µg/L (females); subjects whose SF levels were below these cut-offs were designated as controls. Samples were genotyped for GNPAT p.D519G. We compared the allele frequency of the present subjects, with/without elevated SF, to p.D519G frequency in public datasets. GNPAT p.D519G was more prevalent in our cohort of p.C282Y/p.H63D compound heterozygotes with elevated SF (37%) than European public datasets: 1000G 21%, gnomAD 20% and ESP 21%. We conclude that GNPAT p.D519G is associated with elevated SF in Australian HFE p.C282Y/p.H63D compound heterozygotes.

Phenotypic analysis of hemochromatosis subtypes reveals variations in severity of iron overload and clinical disease.

The clinical progression of HFE-related hereditary hemochromatosis (HH) and its phenotypic variability has been well studied. Less is known about the natural history of non-HFE HH caused by mutations in the HJV, HAMP, or TFR2 genes. The purpose of this study was to compare the phenotypic and clinical presentations of hepcidin-deficient forms of HH. A literature review of all published cases of genetically confirmed HJV, HAMP, and TFR2 HH was performed. Phenotypic and clinical data from a total of 156 patients with non-HFE HH was extracted from 53 publications and compared with data from 984 patients with HFE-p.C282Y homozygous HH from the QIMR Berghofer Hemochromatosis Database. Analyses confirmed that non-HFE forms of HH have an earlier age of onset and a more severe clinical course than HFE HH. HJV and HAMP HH are phenotypically and clinically very similar and have the most severe presentation, with cardiomyopathy and hypogonadism being particularly prevalent findings. TFR2 HH is more intermediate in its age of onset and severity. All clinical outcomes analyzed were more prevalent in the juvenile forms of HH, with the exception of arthritis and arthropathy, which were more commonly seen in HFE HH. This is the first comprehensive analysis comparing the different phenotypic and clinical aspects of the genetic forms of HH, and the results will be valuable for the differential diagnosis and management of these conditions. Importantly, our analyses indicate that factors other than iron overload may be contributing to joint pathology in patients with HFE HH.

Evaluation of a bone morphogenetic protein 6 variant as a cause of iron loading.

BACKGROUND: Atypical iron overload without variation in the five clinically associated hereditary hemochromatosis genes is now recognized; however, their etiology remains unknown. Since the identification of iron overload in the bone morphogenetic protein 6 (Bmp6) knockout mouse, the search has been on for clinically pathogenic variants in the BMP6 gene. A recent report proposes that variants in the pro-peptide region of BMP6 are the underlying cause of several cases of iron overload. We performed targeted next-generation sequencing on three cases of atypical iron overload with Asian ethnicity and identified a p.Q118dup (aka p.E112indelEQ, p.Q115dup, p.Q118_L119insQ) variant in BMP6. The purpose of this study was to characterize the molecular function of the identified BMP6 variant. Molecular characterization by immunofluorescence microscopy and Western blotting of transfected cells, bioinformatics, and population analyses was performed. RESULTS: In contrast to reports for other BMP6 pro-peptide variants in this region, our data indicates that this variant does not affect the function of the mature BMP6 protein. CONCLUSIONS: Our data suggest that assignment of disease causation in clinical cases of iron overload to pro-peptide variants in BMP6 should thus be treated with caution and requires biological characterization.

Cirrhosis in Hemochromatosis: Independent Risk Factors in 368 HFE p.C282Y Homozygotes.

INTRODUCTION AND AIM: We sought to identify independent risk factors for cirrhosis in HFE p.C282Y homozygotes in a cross-sectional study. MATERIAL AND METHODS: We evaluated 368 p.C282Y homozygotes who underwent liver biopsy and compared characteristics of those with and without cirrhosis. We performed multivariable logistic regression on cirrhosis with: age; sex; race/ethnicity; diabetes; blood pints/units donated voluntarily; erythrocyte pints/units received; iron supplement use; alcohol intake, g/d; body mass index, kg/m2; swollen/tender 2nd/3rd metacarpophalangeal joints; elevated alanine aminotransferase; elevated aspartate aminotransferase; steatosis/fatty liver; iron removed by phlebotomy, g; and GNPAT p.D519G positivity. RESULTS: Mean age of 368 participants (73.6% men) was 47 ± 13 (standard deviation) y. Cirrhosis was diagnosed in 86 participants (23.4%). Participants with cirrhosis had significantly greater mean age, proportion of men, diabetes prevalence, mean daily alcohol intake, prevalence of swollen/ tender 2nd/3rd metacarpophalangeal joints, mean serum ferritin, elevated alanine aminotransferase, elevated aspartate aminotransferase, and mean iron removed; and significantly fewer mean blood pints/units donated. GNPAT p.D519G positivity was detected in 82 of 188 participants (43.6%). In a multivariable model for cirrhosis, there were four significant positive associations: age (10-y intervals) (odds ratio 2.2 [95% confidence interval 1.5, 3.3]); diabetes (3.3; [1.1, 9.7]); alcohol intake (14 g alcohol drinks/d) (1.5 [1.2, 1.8]); and iron removed, g (1.3 [1.2, 1.4]). There was no statistical evidence of two-way interactions between these variables. CONCLUSION: In conclusion, cirrhosis in HFE p.C282Y homozygotes is significantly associated with age, diabetes, daily alcohol intake, and iron removed by phlebotomy, taking into account the effect of other variables.

The liver in regulation of iron homeostasis.

The liver is one of the largest and most functionally diverse organs in the human body. In addition to roles in detoxification of xenobiotics, digestion, synthesis of important plasma proteins, gluconeogenesis, lipid metabolism, and storage, the liver also plays a significant role in iron homeostasis. Apart from being the storage site for excess body iron, it also plays a vital role in regulating the amount of iron released into the blood by enterocytes and macrophages. Since iron is essential for many important physiological and molecular processes, it increases the importance of liver in the proper functioning of the body's metabolism. This hepatic iron-regulatory function can be attributed to the expression of many liver-specific or liver-enriched proteins, all of which play an important role in the regulation of iron homeostasis. This review focuses on these proteins and their known roles in the regulation of body iron metabolism.

GNPAT p.D519G is independently associated with markedly increased iron stores in HFE p.C282Y homozygotes.

BACKGROUND: GNPAT p.D519G positivity is significantly increased in HFE p.C282Y homozygotes with markedly increased iron stores. We sought to determine associations of p.D519G and iron-related variables with iron stores in p.C282Y homozygotes. METHODS: We defined markedly increased iron stores as serum ferritin >2247pmol/L (>1000µg/L) and either hepatic iron >236µmol/g dry weight or iron >10g by induction phlebotomy (men and women). We defined normal or mildly elevated iron stores as serum ferritin <674.1pmol/L (<300µg/L) or either age≥40y with iron ≤2.5g iron by induction phlebotomy or age≥50y with ≤3.0g iron by induction phlebotomy (men only). We compared participant subgroups using univariate methods. Using multivariable logistic regression, we evaluated associations of markedly increased iron stores with these variables: age; iron supplement use (dichotomous); whole blood units donated; erythrocyte units received as transfusion; daily alcohol consumption, g; and p.D519G positivity (heterozygosity or homozygosity). RESULTS: The mean age of 56 participants (94.6% men) was 55±10 (SD) y; 41 had markedly increased iron stores. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis. In multivariable analyses, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p=0.0126). CONCLUSIONS: GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and alcohol consumption.

The dynamics of hepcidin-ferroportin internalization and consequences of a novel ferroportin disease mutation.

The hepcidin-ferroportin axis underlies the pathophysiology of many iron-associated disorders and is a key target for the development of therapeutics for treating iron-associated disorders. The aims of this study were to investigate the dynamics of hepcidin-mediated ferroportin internalization and the consequences of a novel disease-causing mutation on ferroportin function. Specific reagents for ferroportin are limited; we developed and characterized antibodies against the largest extracellular loop of ferroportin and developed a novel cell-based assay for studying hepcidin-ferroportin function. We show that hepcidin-mediated ferroportin internalization is a rapid process and could be induced using low concentrations of hepcidin. Targeted next-generation sequencing utilizing an iron metabolism gene panel developed in our group identified a novel ferroportin p.D84E variant in a patient with iron overload. Wild-type and mutant ferroportin constructs were generated, transfected into HEK293 cells and analysed using an all-in-one flow-cytometry-based assay to study the effects on hepcidin-mediated internalization and iron transport. Consistent with the classical phenotype of ferroportin disease, the p.D84E mutation results in an inability to transport iron and hepcidin insensitivity. These results validate a recently proposed 3D-structural model of ferroportin and highlight the significance of this variant in the structure and function of ferroportin. Our novel ferroportin antibody and assay will be valuable tools for investigating the regulation of hepcidin/ferroportin function and the development of novel approaches for the therapeutic modulation of iron homeostasis.

Normal systemic iron homeostasis in mice with macrophage-specific deletion of transferrin receptor 2.

Iron is an essential element, since it is a component of many macromolecules involved in diverse physiological and cellular functions, including oxygen transport, cellular growth, and metabolism. Systemic iron homeostasis is predominantly regulated by the liver through the iron regulatory hormone hepcidin. Hepcidin expression is itself regulated by a number of proteins, including transferrin receptor 2 (TFR2). TFR2 has been shown to be expressed in the liver, bone marrow, macrophages, and peripheral blood mononuclear cells. Studies from our laboratory have shown that mice with a hepatocyte-specific deletion of Tfr2 recapitulate the hemochromatosis phenotype of the global Tfr2 knockout mice, suggesting that the hepatic expression of TFR2 is important in systemic iron homeostasis. It is unclear how TFR2 in macrophages contributes to the regulation of iron metabolism. We examined the role of TFR2 in macrophages by analysis of transgenic mice lacking Tfr2 in macrophages by crossing Tfr2(f/f) mice with LysM-Cre mice. Mice were fed an iron-rich diet or injected with lipopolysaccharide to examine the role of macrophage Tfr2 in iron- or inflammation-mediated regulation of hepcidin. Body iron homeostasis was unaffected in the knockout mice, suggesting that macrophage TFR2 is not required for the regulation of systemic iron metabolism. However, peritoneal macrophages of knockout mice had significantly lower levels of ferroportin mRNA and protein, suggesting that TFR2 may be involved in regulating ferroportin levels in macrophages. These studies further elucidate the role of TFR2 in the regulation of iron homeostasis and its role in regulation of ferroportin and thus macrophage iron homeostasis.

The global prevalence of HFE and non-HFE hemochromatosis estimated from analysis of next-generation sequencing data.

PURPOSE: The prevalence of HFE-related hereditary hemochromatosis (HH) among European populations has been well studied. There are no prevalence data for atypical forms of HH caused by mutations in HFE2, HAMP, TFR2, or SLC40A1. The purpose of this study was to estimate the population prevalence of these non-HFE forms of HH. METHODS: A list of HH pathogenic variants in publically available next-generation sequence (NGS) databases was compiled and allele frequencies were determined. RESULTS: Of 161 variants previously associated with HH, 43 were represented among the NGS data sets; an additional 40 unreported functional variants also were identified. The predicted prevalence of HFE HH and the p.Cys282Tyr mutation closely matched previous estimates from similar populations. Of the non-HFE forms of iron overload, TFR2-, HFE2-, and HAMP-related forms are predicted to be rare, with pathogenic allele frequencies in the range of 0.00007 to 0.0005. Significantly, SLC40A1 variants that have been previously associated with autosomal-dominant ferroportin disease were identified in several populations (pathogenic allele frequency 0.0004), being most prevalent among Africans. CONCLUSION: We have, for the first time, estimated the population prevalence of non-HFE HH. This methodology could be applied to estimate the population prevalence of a wide variety of genetic disorders.Genet Med 18 6, 618-626.

Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload.

UNLABELLED: To identify polymorphisms associated with variability of iron overload severity in HFE-associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n = 22; cases) or with normal or mildly increased iron stores (n = 13; controls). The 35 participants, residents of the United States, Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single-nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload (P = 3 × 10(-6) ; P = 0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of twenty-two participants with severe iron overload had glyceronephosphate O-acyltransferase (GNPAT) polymorphism p.D519G (rs11558492; 15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed small interfering RNA-based knockdown of GNPAT in the human liver-derived cell line, HepG2/C3A. This knockdown resulted in a >17-fold decrease in expression of the messenger RNA encoding the iron-regulatory hormone, hepcidin. CONCLUSION: GNPAT p.D519G is associated with a high-iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation.

Analysis of IL-22 contribution to hepcidin induction and hypoferremia during the response to LPS in vivo.

The anaemia of chronic disease (ACD) results from inflammation-mediated up-regulation of the iron regulatory hormone hepcidin, with the consequent sequestration of iron limiting its availability for erythropoiesis. The inflammatory cytokine IL-6, a regulator of hepcidin, has been implicated in this process. Recent in vivo and in vitro studies indicate that IL-22 is also able to stimulate hepcidin expression. We aimed to determine if IL-22 had a role in causing the hypoferremia associated with the inflammatory response. Wild-type and Il22-knockout mice were subjected to an acute inflammatory stimulus via administration of LPS and the response of hepcidin and iron homeostasis was analysed. In the absence of IL-22, there was a response of hepcidin, resulting in a reduction in serum iron levels. However, the hypoferremic response to LPS was slightly blunted in mice lacking IL-22, suggesting that, during LPS-mediated inflammation, IL-22 may play a minor role in mediating the hypoferremic response. These results may have implications for the treatment and management of the ACD.

Hematopoietic deletion of transferrin receptor 2 in mice leads to a block in erythroid differentiation during iron-deficient anemia.

Iron metabolism and erythropoiesis are inherently interlinked physiological processes. Regulation of iron metabolism is mediated by the iron-regulatory hormone hepcidin. Hepcidin limits the amount of iron released into the blood by binding to and causing the internalization of the iron exporter, ferroportin. A number of molecules and physiological stimuli, including erythropoiesis, are known to regulate hepcidin. An increase in erythropoietic demand decreases hepcidin, resulting in increased bioavailable iron in the blood. Transferrin receptor 2 (TFR2) is involved in the systemic regulation of iron metabolism. Patients and mice with mutations in TFR2 develop hemochromatosis due to inappropriate hepcidin levels relative to body iron. Recent studies from our laboratory and others have suggested an additional role for TFR2 in response to iron-restricted erythropoiesis. These studies used mouse models with perturbed systemic iron metabolism: anemic mice lacking matriptase-2 and Tfr2, or bone marrow transplants from iron-loaded Tfr2 null mice. We developed a novel transgenic mouse model which lacks Tfr2 in the hematopoietic compartment, enabling the delineation of the role of Tfr2 in erythroid development without interfering with its role in systemic iron metabolism. We show that in the absence of hematopoietic Tfr2 immature polychromatic erythroblasts accumulate with a concordant reduction in the percentage of mature erythroid cells in the spleen and bone marrow of anemic mice. These results demonstrate that erythroid Tfr2 is essential for an appropriate erythropoietic response in iron-deficient anemia. These findings may be of relevance in clinical situations in which an immediate and efficient erythropoietic response is required. Am. J. Hematol. 91:812-818, 2016. © 2016 Wiley Periodicals, Inc.

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

Next-generation sequencing: Application of a novel platform to analyze atypical iron disorders.

The development of targeted next-generation sequencing (NGS) applications now promises to be a clinically viable option for the diagnosis of rare disorders. This approach is proving to have significant utility where standardized testing has failed to identify the underlying molecular basis of disease. We have developed a unique targeted NGS panel for the systematic sequence-based analysis of atypical iron disorders. We report the analysis of 39 genes associated with iron regulation in eight cases of atypical iron dysregulation, in which five cases we identified the definitive causative mutation, and a possible causative mutation in a sixth. We further provide a molecular and cellular characterization study of one of these mutations (TFR2, p.I529N) in a familial case as proof of principle. Cellular analysis of the mutant protein indicates that this amino acid substitution affects the localization of the protein, which results in its retention in the endoplasmic reticulum and thus failure to function at the cell surface. Our unique NGS panel presents a rapid and cost-efficient approach to identify the underlying genetic cause in cases of atypical iron homeostasis disorders.

A critical role for murine transferrin receptor 2 in erythropoiesis during iron restriction.

Effective erythropoiesis requires an appropriate supply of iron and mechanisms regulating iron homeostasis and erythropoiesis are intrinsically linked. Iron dysregulation, typified by iron-deficiency anaemia and iron overload, is common in many clinical conditions and impacts the health of up to 30% of the world's population. The proteins transmembrane protease, serine 6 (TMPRSS6; also termed matriptase-2), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis, by regulating expression of the iron regulatory hormone hepcidin. We have performed a systematic analysis of mice deficient in these three proteins and show that TMPRSS6 predominates over HFE and TFR2 in hepcidin regulation. The phenotype of mice lacking TMPRSS6 and TFR2 is characterized by severe anaemia and extramedullary haematopoiesis in the spleen. Stress erythropoiesis in these mice results in increased expression of the newly identified erythroid iron regulator erythroferrone, which does not appear to overcome the hepcidin overproduction mediated by loss of TMPRSS6. Extended analysis reveals that TFR2 plays an important role in erythroid cells, where it is involved in terminal erythroblast differentiation and the regulation of erythropoietin. In conclusion, we have identified an essential role for TFR2 in erythropoiesis that may provide new targets for the treatment of anaemia.

Inconsistent hepatic antifibrotic effects with the iron chelator deferasirox.

BACKGROUND AND AIM: Development of effective antifibrotic treatments that can be translated to clinical practice is an important challenge in contemporary hepatology. A recent report on ß-thalassemia patients demonstrated that deferasirox treatment reversed or stabilized liver fibrosis independent of its iron-chelating properties. In this study, we investigated deferasirox in cell and animal models to better understand its potential antifibrotic effects. METHODS: The LX-2 stellate cell line was treated with 5 µM or 50 µM deferasirox (Exjade, Novartis Pharmaceuticals Australia, North Ryde, NSW, Australia) for up to 120 h. Three-week-old multidrug resistance 2 null (Mdr2(-/-) ) mice received oral deferasirox or vehicle for 4 weeks (30 mg/kg/day). Cells and liver tissue were collected for assessment of fibrosis and fibrogenic gene expression. RESULTS: In LX-2 cells treated with 50 µM deferasirox for 12 h, α1(I)procollagen expression was decreased by 25%, with maximal reductions (10-fold) seen following 24-120 h of treatment. Similarly, α-smooth muscle actin (αSMA) expression was significantly lower. Alterations in matrix remodeling genes, specifically decreased expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2, were observed. There was no significant difference in hepatic hydroxyproline content in Mdr2(-/-) mice following deferasirox administration (vehicle: 395 ± 27 µg/g vs deferasirox: 421 ± 33 µg/g). Similarly, no changes in the expression of fibrogenic genes were observed. CONCLUSION: Despite reductions in α1(I)procollagen and αSMA expression and alterations in matrix degradation genes in LX-2 cells, deferasirox did not exhibit antifibrotic activity in Mdr2(-/-) mice. Given the positive outcomes seen in human trials, it may be appropriate to study deferasirox in other animal models of fibrosis and/or for a longer duration of therapy.

Parenteral vs. oral iron: influence on hepcidin signaling pathways through analysis of Hfe/Tfr2-null mice.

Treatment for iron deficiency anemia can involve iron supplementation via dietary or parenteral routes that result in different cellular iron distributions. The effect of the administered iron on the iron regulatory system and hepcidin in the liver has not been well studied. Hepcidin, the liver-expressed central iron-regulatory peptide, is itself regulated through the bone morphogenetic protein (BMP)/SMAD signaling pathway. Specifically, Bmp6 expression is upregulated in response to iron and induces hepcidin through phosphorylation of Smad1/5/8. The hemochromatosis-associated proteins Hfe and transferrin receptor 2 (Tfr2) are known upstream regulators of hepcidin, although their precise roles are still unclear. To investigate the mechanisms of this regulation and the roles of the Hfe and Tfr2, we subjected wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice to iron loading via dietary or parenteral routes. Systematic analysis demonstrated that Tfr2 is required for effective upregulation of Bmp6 in response to hepatocyte iron, but not nonparenchymal iron. Hfe is not required for Bmp6 upregulation, regardless of iron localization, but rather, is required for efficient downstream transmission of the regulatory signal. Our results demonstrate that Hfe and Tfr2 play separate roles in the regulatory responses to iron compartmentalized in different cell types and further elucidates the regulatory mechanisms controlling iron homeostasis.