Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.
Hepcidin is a peptide hormone secreted by the liver that plays a central role in the regulation of iron homeostasis. Increased hepcidin levels result in anemia while decreased expression is the causative feature in most primary iron overload diseases. Mutations in hemochromatosis type 2 (HFE2), which encodes the protein hemojuvelin (HJV), result in the absence of hepcidin and an early-onset form of iron overload disease. HJV is a bone morphogenetic protein (BMP) coreceptor and HJV mutants have impaired BMP signaling. In this issue of the JCI, Babitt and colleagues show that BMPs are autocrine hormones that induce hepcidin expression (see the related article beginning on page 1933). Administration of a recombinant, soluble form of HJV decreased hepcidin expression and increased serum iron levels by mobilizing iron from splenic stores. These results demonstrate that recombinant HJV may be a useful therapeutic agent for treatment of the anemia of chronic disease, a disorder resulting from high levels of hepcidin expression.
Hemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE, transferrin receptor 2, ferroportin, hepcidin, and hemojuvelin (HJV). Recent studies have established the expression of all the five genes in retina, indicating their importance in retinal iron homeostasis. We previously demonstrated that Hjv is expressed in retinal pigment epithelium (RPE), outer and inner nuclear layers, and ganglion cell layer. Here we report on the consequences of Hjv deletion on the retina in mice. Hjv-null mice at ≥ 18 months of age had increased iron accumulation in retina with marked morphological damage compared to age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-null mice included hyperplasia of RPE. We isolated RPE cells from wild type and Hjv-null mice and examined their growth patterns. Hjv-null RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-null RPE cells also showed upregulation of Slc7a11 that codes for the ‘transporter proper’ subunit xCT in the heterodimeric amino acid transporter cystine/glutamate exchanger (xCT/4F2hc). Bone morphogenic protein (BMP) 6 could not induce hepcidin expression in Hjv-null RPE cells, confirming that retinal cells require Hjv for induction of hepcidin via BMP6 signaling. Hjv is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated Hjv is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these studies confirm the biological importance of Hjv in the regulation of iron homeostasis in the retina and in RPE.
hemojuvelin; hemochromatosis; retinal pigment epithelium; hepcidin; bone morphogenic protein; knockout mouse
Hereditary hemochromatosis is an iron-overload disorder resulting from mutations in proteins presumed to be involved in the maintenance of iron homeostasis. Mutations in hemojuvelin (HJV) cause severe, early-onset juvenile hemochromatosis. The normal function of HJV is unknown. Juvenile hemochromatosis patients have decreased urinary levels of hepcidin, a peptide hormone that binds to the cellular iron exporter ferroportin, causing its internalization and degradation. We have disrupted the murine Hjv gene and shown that Hjv–/– mice have markedly increased iron deposition in liver, pancreas, and heart but decreased iron levels in tissue macrophages. Hepcidin mRNA expression was decreased in Hjv–/– mice. Accordingly, ferroportin expression detected by immunohistochemistry was markedly increased in both intestinal epithelial cells and macrophages. We propose that excess, unregulated ferroportin activity in these cell types leads to the increased intestinal iron absorption and plasma iron levels characteristic of the juvenile hemochromatosis phenotype.
Iron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV) is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis). Currently however, there is no information on how HJV is regulated by inflammation.
To study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7) were used to study the effect of IL-6 and TNF-α on HJV mRNA expression.
Here we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL) decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc), we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE) within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional.
From these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.
Inflammation; Hemojuvelin; TNF-α
Juvenile hemochromatosis is an iron overload disorder caused by mutations in the genes encoding the major iron regulatory hormone hepcidin (HAMP)1 and hemojuvelin (HFE2)2. We have previously shown that hemojuvelin is a bone morphogenetic protein (BMP) co-receptor and that BMP signals regulate hepcidin expression and iron metabolism3,4. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here, we show that in vitro, compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc more potently inhibits hepcidin induction by BMP-2 or BMP-4, but less potently inhibits BMP-6. In vivo, HJV.Fc or a neutralizing BMP-6 antibody inhibits hepcidin expression and increases serum iron, while DRAGON.Fc has no effect. Notably, Bmp6 null mice have a phenotype resembling hereditary hemochromatosis with reduced hepcidin expression and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP-6, and we show that BMP-6 increases hepcidin expression and reduces serum iron in mice. These data support a key role for BMP-6 as a ligand for HJV and an endogenous regulator of hepcidin expression and iron metabolism in vivo.
A 55 year old man with a history of chronic hepatic C infection was found to have severe hemochromatosis: hepatic cirrhosis, cardiomyopathy, arrhythmia, hypogonadism, diabetes and bronzed skin color. After 50 phlebotomies, he underwent a combined heart and liver transplant. Genetic analyses identified a novel mutation in the iron responsive element of the ALAS2 gene. No mutations were found in other genes associated with adult or juvenile hemochromatosis including HFE, transferrin receptor-2 (TfR2), ferroportin (SLC40A1), hepcidin (HAMP) and hemojuvelin (HJV). We suggest that the ALAS2 mutation together with chronic hepatitis C infection may have caused the severe iron overload phenotype.
ALAS2; iron responsive element; iron overload; hepatitis C; liver transplant; hemochromatosis
Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by enhanced intestinal absorption of dietary iron. Without therapeutic intervention, iron overload leads to multiple organ damage such as liver cirrhosis, cardiomyopathy, diabetes, arthritis, hypogonadism and skin pigmentation. Most HH patients carry HFE mutant genotypes: homozygosity for p.Cys282Tyr or p.Cys282Tyr/p.His63Asp compound heterozygosity. In addition to HFE gene, mutations in the genes that encode hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) have been associated with regulation of iron homeostasis and development of HH. The aim of this review was to identify the main gene mutations involved in the pathogenesis of type 1, 2, 3 and 4 HH and their genetic testing indication. HFE testing for the two main mutations (p.Cys282Tyr and p.His63Asp) should be performed in all patients with primary iron overload and unexplained increased transferrin saturation and/or serum ferritin values. The evaluation of the HJV p.Gly320Val mutation must be the molecular test of choice in suspected patients with juvenile hemochromatosis with less than 30 years and cardiac or endocrine manifestations. In conclusion, HH is an example that genetic testing can, in addition to performing the differential diagnostic with secondary iron overload, lead to more adequate and faster treatment.
hemochromatosis; primary iron overload; HFE; high-resolution melting; HJV; molecular diagnostic
Hereditary hemochromatosis (HH) encompasses genetic disorders of iron overload characterized by deficient expression or function of the iron-regulatory hormone hepcidin. Mutations in 5 genes have been linked to this disease: HFE, TFR2 (encoding transferrin receptor 2), HAMP (encoding hepcidin), SLC40A1 (encoding ferroportin) and HJV (encoding hemojuvelin). Hepcidin inhibits iron export from cells into plasma. Hemojuvelin, an upstream regulator of hepcidin expression, is expressed in mice mainly in the heart and skeletal muscle. It has been suggested that soluble hemojuvelin shed by the muscle might reach the liver to influence hepcidin expression. Heart muscle is one of the target tissues affected by iron overload, with resultant cardiomyopathy in some HH patients. Therefore, we investigated the effect of iron overload on gene expression in skeletal muscle and heart using Illumina™ arrays containing over 47,000 probes. The most apparent changes in gene expression were confirmed using real-time RT-PCR.
Genes with up-regulated expression after iron overload in both skeletal and heart muscle included angiopoietin-like 4, pyruvate dehydrogenase kinase 4 and calgranulin A and B. The expression of transferrin receptor, heat shock protein 1B and DnaJ homolog B1 were down-regulated by iron in both muscle types. Two potential hepcidin regulatory genes, hemojuvelin and neogenin, showed no clear change in expression after iron overload.
Microarray analysis revealed iron-induced changes in the expression of several genes involved in the regulation of glucose and lipid metabolism, transcription and cellular stress responses. These may represent novel connections between iron overload and pathological manifestations of HH such as cardiomyopathy and diabetes.
In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV) or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin mRNA responsiveness to short and long-term iron challenge in iron-depleted Hfe, Tfr2, Hjv and Bmp6 mutant mice. After 1-day (acute) iron challenge, Hfe−/− showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6−/− nearly no increase, and Tfr2 and Hjv mutants no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21-day (chronic) iron challenge, Hfe and Tfr2 mutants increased hepcidin expression to nearly wild-type levels but a blunted increase of hepcidin was seen in Bmp6−/− and Hjv−/− mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (excepting Bmp6−/− mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV and BMP6 and, to a lesser extent, HFE, are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading, and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2 and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading.
Hereditary hemochromatosis; bone morphogenetic protein 6; hemojuvelin; HFE; transferrin receptor 2
Recently, mutations causing juvenile hemochromatosis have been identified in a novel gene, hemojuvelin (HJV), located on chromosome 1. Mouse models of this disease have now been developed by 2 groups, Huang et al. and Niederkofler et al., through targeted disruption of the Hjv gene (see the related articles beginning on pages 2180 and 2187). These mutant mice will allow further investigation into the role of HJV in the regulation of iron homeostasis, a role that to date remains elusive.
Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl4. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.
Hemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency.
C57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv−/− mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit.
Liver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv−/− mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA.
Since no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.
Hereditary hemochromatosis (HH) is an autosomal recessive disorder classically related to HFE mutations. However, since 1996, it is known that HFE mutations explain about 80% of HH cases, with the remaining around 20% denominated non-HFE hemochromatosis. Nowadays, four main genes are implicated in the pathophysiology of clinical syndromes classified as non-HFE hemochromatosis: hemojuvelin (HJV, type 2Ajuvenile HH), hepcidin (HAMP, type 2B juvenile HH), transferrin receptor 2 (TFR2, type 3 HH) and ferroportin (SLC40A1, type 4 HH). The aim of this review is to explore molecular, clinical and management aspects of non-HFE hemochromatosis.
Hemochromatosis; Iron overload; Iron metabolism disorders
There are few descriptions of young adults with self-reported hemochromatosis or iron overload (H/IO). We analyzed initial screening data in 7,343 HEmochromatosis and IRon Overload Screening (HEIRS) Study participants ages 25–29 years, including race/ethnicity and health information; transferrin saturation (TS) and ferritin (SF) measurements; and HFE C282Y and H63D genotypes. We used denaturing high-pressure liquid chromatography and sequencing to detect mutations in HJV, TFR2, HAMP, SLC40A1, and FTL. Fifty-one participants reported previous H/IO; 23 (45%) reported medical conditions associated with H/IO. Prevalences of reports of arthritis, diabetes, liver disease or liver cancer, heart failure, fertility problems or impotence, and blood relatives with H/IO were significantly greater in participants with previous H/IO reports than in those without. Only 7.8% of the 51 participants with previous H/IO reports had elevated TS; 13.7% had elevated SF. Only one participant had C282Y homozygosity. Three participants aged 25–29 years were heterozygous for potentially deleterious mutations in HFE2, TFR2, and HAMP promoter, respectively. Prevalences of self-reported conditions, screening iron phenotypes, and C282Y homozygosity were similar in 1,165 participants aged 30 years or greater who reported previous H/IO. We conclude that persons who report previous H/IO diagnoses in screening programs are unlikely to have H/IO phenotypes or genotypes. Previous H/IO reports in some participants could be explained by treatment that induced iron depletion before initial screening, misdiagnosis, or participant misunderstanding of their physician or the initial screening questionnaire.
Hereditary hemochromatosis (HFE), which affects 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals in Western population, results in multiple organ damage caused by iron deposition, and is treatable if detected early. C282Y mutation in HFE gene has been known to be responsible for the most hereditary hemochromatosis cases and 5-10% of white subjects are heterozygous for this mutation. However, the prevalence of hemochromatosis in the Asian population was reported to be very low and ethnic heterogeneity has been suspected. The aim of our study was to determine the prevalence of heterozygosity and homozygosity for the C282Y HFE gene mutations in 502 unrelated Koreans. Results revealed that none of them had the mutant gene, suggesting a significant ethnic difference when compared with Caucasians. Our study excluded underlying possibility of hereditary hemochromatosis in Korean which could mimic the findings of alcoholic liver disease with iron overload or liver cirrhosis with chronic hepatitis C.
Systemic iron balance is regulated by hepcidin, a peptide hormone secreted by the liver. By decreasing cell surface expression of the iron exporter ferroportin, hepcidin decreases iron absorption from the intestine and iron release from reticuloendothelial stores. Hepcidin excess has been implicated in the pathogenesis of anemia of chronic disease, while hepcidin deficiency has a key role in the pathogenesis of the iron overload disorder hemochromatosis. We have recently shown that hemojuvelin is a coreceptor for bone morphogenetic protein (BMP) signaling and that BMP signaling positively regulates hepcidin expression in liver cells in vitro. Here we show that BMP-2 administration increases hepcidin expression and decreases serum iron levels in vivo. We also show that soluble hemojuvelin (HJV.Fc) selectively inhibits BMP induction of hepcidin expression in vitro and that administration of HJV.Fc decreases hepcidin expression, increases ferroportin expression, mobilizes splenic iron stores, and increases serum iron levels in vivo. These data support a role for modulators of the BMP signaling pathway in treating diseases of iron overload and anemia of chronic disease.
Mutations in the hepcidin gene HAMP and the hemojuvelin gene HJV have recently been shown to result in juvenile haemochromatosis (JH). Hepcidin is an antimicrobial peptide that plays a key role in regulating intestinal iron absorption. Hepcidin levels are reduced in patients with haemochromatosis due to mutations in the HFE and HJV genes. Digenic inheritance of mutations in HFE and HAMP can result in either JH or hereditary haemochromatosis (HH) depending upon the severity of the mutation in HAMP. Here we review these findings and discuss how understanding the different types of haemochromatosis and our increasing knowledge of iron metabolism may help to elucidate the host's response to infection.
In age-matched cohorts of screening study participants recruited from primary care clinics, mean serum transferrin saturation values were significantly lower and mean serum ferritin concentrations were significantly higher in Native Americans than in whites. Twenty-eight percent of 80 Alabama white hemochromatosis probands with HFE C282Y homozygosity previously reported having Native American ancestry, but the possible effect of this ancestry on hemochromatosis phenotypes was unknown.
We compiled observations in these 80 probands and used univariate and multivariate methods to analyze associations of age, sex, Native American ancestry (as a dichotomous variable), report of ethanol consumption (as a dichotomous variable), percentage transferrin saturation and loge serum ferritin concentration at diagnosis, quantities of iron removed by phlebotomy to achieve iron depletion, and quantities of excess iron removed by phlebotomy.
In a univariate analysis in which probands were grouped by sex, there were no significant differences in reports of ethanol consumption, transferrin saturation, loge serum ferritin concentration, quantities of iron removed to achieve iron depletion, and quantities of excess iron removed by phlebotomy in probands who reported Native American ancestry than in those who did not. In multivariate analyses, transferrin saturation (as a dependent variable) was not significantly associated with any of the available variables, including reports of Native American ancestry and ethanol consumption. The independent variable quantities of excess iron removed by phlebotomy was significantly associated with loge serum ferritin used as a dependent variable (p < 0.0001), but not with reports of Native American ancestry or reports of ethanol consumption. Loge serum ferritin was the only independent variable significantly associated with quantities of excess iron removed by phlebotomy used as a dependent variable (p < 0.0001) (p < 0.0001; ANOVA of regression).
We conclude that the iron-related phenotypes of hemochromatosis probands with HFE C282Y homozygosity are similar in those with and without Native American ancestry reports.
pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv−/−) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv−/− mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv−/− mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.
The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation and downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2MASK), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2MASK shows no and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2MASK. The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.
Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.
Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass.
We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant.
Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.
Iron is essential for cell survival and function; yet excess iron is toxic to cells. Therefore, the cellular and whole-body levels of iron are regulated exquisitely. At least a dozen proteins participate in the regulation of iron homeostasis. Hemochromatosis, a genetic disorder of iron overload, is caused by mutations in at least five genes, namely HFE, hemojuvelin, Transferrin receptor 2, ferroportin, and hepcidin. Retina is separated from systemic circulation by inner and outer blood-retinal barriers; therefore it is widely believed that this tissue is immune to changes in systemic circulation. Even though hemochromatosis is associated with iron overload and dysfunction of a variety of systemic organs, little is known on the effects of this disease on the retina. Recent studies have shown that all five genes that are associated with hemochromatosis are expressed in the retina in a cell type-specific manner. The retinal pigment epithelium, which forms the outer blood-retinal barrier, expresses all of these five genes. It is therefore clearly evident that iron homeostasis in the retina is maintained locally by active participation of various iron-regulatory proteins. Excess iron is detrimental to the retina as evidenced from human studies and from mouse models of iron overload. Retinal iron homeostasis is disrupted in various clinical conditions such as hemochromatosis, aceruloplasminemia, age-related macular degeneration, and bacterial and viral infections.
Hemochromatosis; age-related macular degeneration; iron toxicity; oxidative damage; retina
Hereditary Hemochromatosis (HH) is a recessively inherited disorder of iron overload occurring commonly in subjects homozygous for the C282Y mutation in HFE gene localized on chromosome 6p21.3 in linkage disequilibrium with the human leukocyte antigen (HLA)-A locus. Although its genetic homogeneity, the phenotypic expression is variable suggesting the presence of modifying factors. One such genetic factor, a SNP microhaplotype named A-A-T, was recently found to be associated with a more severe phenotype and also with low CD8+T-lymphocyte numbers. The present study aimed to test whether the predictive value of the A-A-T microhaplotype remained in other population settings. In this study of 304 HH patients from 3 geographically distant populations (Porto, Portugal 65; Alabama, USA 57; Nord-Trøndelag, Norway 182), the extended haplotypes involving A-A-T were studied in 608 chromosomes and the CD8+ T-lymphocyte numbers were determined in all subjects. Patients from Porto had a more severe phenotype than those from other settings. Patients with A-A-T seemed on average to have greater iron stores (p = 0.021), but significant differences were not confirmed in the 3 separate populations. Low CD8+ T-lymphocytes were associated with HLA-A*03-A-A-T in Porto and Alabama patients but not in the greater series from Nord-Trøndelag. Although A-A-T may signal a more severe iron phenotype, this study was unable to prove such an association in all population settings, precluding its use as a universal predictive marker of iron overload in HH. Interestingly, the association between A-A-T and CD8+ T-lymphocytes, which was confirmed in Porto and Alabama patients, was not observed in Nord-Trøndelag patients, showing that common HLA haplotypes like A*01–B*08 or A*03–B*07 segregating with HFE/C282Y in the three populations may carry different messages. These findings further strengthen the relevance of HH as a good disease model to search for novel candidate loci associated with the genetic transmission of CD8+ T-lymphocyte numbers.