Hemojuvelin Knockout Mice Research Articles

The functional role of hemojuvelin in acute ischemic stroke

Our study aimed to establish the role of hemojuvelin (HJV) in acute ischemic stroke (AIS). We performed immunohistochemistry for HJV expression in human brain tissues from 10 AIS and 2 non-stroke autopsy subjects. Plasma HJV was measured in 112 AIS patients within 48 h after stroke. The results showed significantly increased HJV expression in brain tissues from AIS patients compare to non-stroke subjects. After adjusting for clinical variables, plasma levels of HJV within 48 h after stroke were an independent predictor of poor functional outcome three months post-stroke (OR:1.78, 95% CI: 1.03–3.07; P = 0.038). In basic part, Western blotting showed that HJV expression in mice brains was apparent at 3 h after middle cerebral artery occlusion (MCAO), and increased significantly at 72 h. In cultured cortical neurons, expression of HJV protein increased remarkably 24 h after oxygen glucose deprivation (OGD), and small interfering RNAs (siHJV) transfected OGD neurons had a lower apoptotic rate. Importantly, 72 h post-MCAO, HJV knockout mice had significantly smaller infarcts and less expression of cleaved caspase-3 protein compared with wild-type mice. In summary, HJV participates in the mechanisms of post-stroke neuronal injury, and that plasma HJV levels can be a potential early outcome indicator for AIS patients.

Abstract WMP75: Functional Role of Hemojuvelin in Acute Ischemic Stroke

Background and Purpose: Hemojuvelin (HJV)-hepcidin axis has been shown an important pathophysiological mechanism in acute liver and kidney injuries, but little is known about the role of HJV in acute stroke. Our aim was to establish the temporal expression and function of HJV during acute ischemic stroke (AIS). Methods: C57BL/6J mice receiving 1-hour middle cerebral artery occlusion (MCAO) and primary cortical neurons subjected to oxygen and glucose deprivation (OGD) were performed to determine the expression profile of HJV after AIS. Infarct sizes and behavior scores at 72 hours post MCAO on HJV knockout (129S-Hfe2tm1Nca/J) and wild type (129S) mice were compared to determine the role of HJV. In addition, patients with AIS were recruited and plasma samples were collected for the measurement of HJV. Results: The expression of HJV protein in mice brain was noted early at 3 hours after MCAO and increased significantly at 72 hours after MCAO, along with the appearance of its downstream protein- hepcidin at the same time point. In cultured cortical neurons, the expression of HJV protein also increased remarkably at 24-48 hours after 3 hours OGD stimulation. Importantly, HJV knockout mice had significantly less infarct size and expression of cleaved caspase-3 protein compared with wild type mice at 72 hours post MCAO. In human subjects, HJV expression can be seen in autopsy brain tissues from stroke patients with mortality outcome. In addition, among 112 AIS patients, plasma level of HJV within 48 hours after stroke was an independent poor prognostic predictor of functional outcome at 3 months post-stroke (odds ratio: 1.780; 95% confidence intervals: 1.032-3.073; p =0.038). Conclusions: HJV- hepcidin axis is existing in mice brain and participating in the mechanisms of post stroke neuronal injury. Clinically, plasma HJV may be a potential outcome indicator in patients with AIS.

Advanced iron-overload cardiomyopathy in a genetic murine model is rescued by resveratrol therapy.

Iron-overload cardiomyopathy is prevalent on a worldwide basis and is a major comorbidity in patients with genetic hemochromatosis and secondary iron overload. Therapies are limited in part due to lack of a valid preclinical model, which recapitulates advanced iron-overload cardiomyopathy. Male hemojuvelin (HJV) knockout (HJVKO) mice, which lack HJV, a bone morphogenetic co-receptor protein required for hepcidin expression and systemic iron homeostasis, were fed a high-iron diet starting at 4 weeks of age for a duration of 1 year. Aged HJVKO mice in response to iron overload showed increased myocardial iron deposition and mortality coupled with oxidative stress and myocardial fibrosis culminating in advanced iron-overload cardiomyopathy. In a parallel group, iron-overloaded HJVKO mice received resveratrol (240 mg/day) at 9 months of age until 1 year of age. Echocardiography and invasive pressure–volume (PV) loop analyses revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. In addition, myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCa2a) levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCa2a levels and suppressed up-regulation of the sodium–calcium exchanger (NCX1). Further, iron-mediated oxidative stress and myocardial fibrosis were suppressed by resveratrol treatment with concomitant activation of the p-Akt and p-AMP-activated protein kinase (AMPK) signaling pathways. A combination of ageing and high-iron diet in male HJVKO mice results in a valid preclinical model that recapitulates iron-overload cardiomyopathy in humans. Resveratrol therapy resulted in normalization of cardiac function demonstrating that resveratrol represents a feasible therapeutic intervention to reduce the burden of iron-overload cardiomyopathy.

Nicotinamide N-Methyltransferase Expression Decreases in Iron Overload Exacerbating Iron-Induced Hepatotoxicity

Iron overload causes the generation of reactive oxygen species, which can lead to liver failure, particularly when it occurs in conjunction with nonalcoholic fatty liver disease (NAFLD) or viral hepatitis. Previously, we studied the effects of iron deficiency and iron overload on the hepatic transcriptional and metabolomic profile in mice. We found that iron overload induced significant changes in the expression of genes and metabolites involved in glucose and nicotinamide metabolism in both a dietary model, high iron diet, and a genetic model, hemojuvelin knockout mice. In particular, we discovered that the transcript, Nicotinamide N-methyltransferase (NNMT), was significantly downregulated in both models of iron overload. NNMT methylates nicotinamide, a form of vitamin B3, and has recently been shown to play a critical role in hepatic glucose and cholesterol metabolism by stabilizing Sirtuin 1, a nicotinamide-dependent deacetylase. For the current study, we evaluated 63 morbidly obese human subjects who gave written informed consent, validated and approved by the ethical committee of the Hospital Universitari Dr. Josep Trueta (Comitè d’Ètica d’Investigació Clínica, approval number 2009046), for the collection of liver biopsy samples and sera at the time of bariatric surgery after 8 hours of fasting. In these subjects, we found that log hepatic transcript levels of NNMT negatively correlated with measures of body iron stores, including log serum ferritin levels (R=-0.39, p=0.002) and log percent transferrin saturation (R=-0.21, p=0.02). We then performed experiments in primary hepatocytes to evaluate the interaction of iron overload and NNMT. We found that treatment with ferric ammonium citrate significantly decreased NNMT transcript levels in either primary human or mouse hepatocytes. We manipulated NNMT expression in primary mouse hepatocytes using adenoviral vectors and discovered that NNMT knockdown exacerbated iron-induced cytotoxicity and enhanced iron-induced expression of transcriptional markers of oxidative and endoplasmic reticulum stress, while NNMT overexpression protected against iron-induced cytotoxicity. In future studies, we would like to elucidate the mechanisms by which NNMT modulates hepatocyte sensitivity to iron overload. A deeper understanding of the relationship of the pathways involved in maintaining iron and energy homeostasis may reveal new therapeutic targets for patients with iron overload disorders, obesity, and NAFLD. DisclosuresNo relevant conflicts of interest to declare.

Hereditary Hemochromatosis Predisposes Mice to Yersinia pseudotuberculosis Infection Even in the Absence of the Type III Secretion System.

The iron overload disorder hereditary hemochromatosis (HH) predisposes humans to serious disseminated infection with pathogenic Yersinia as well as several other pathogens. Recently, we showed that the iron-sulfur cluster coordinating transcription factor IscR is required for type III secretion in Y. pseudotuberculosis by direct control of the T3SS master regulator LcrF. In E. coli and Yersinia, IscR levels are predicted to be regulated by iron bioavailability, oxygen tension, and oxidative stress, such that iron depletion should lead to increased IscR levels. To investigate how host iron overload influences Y. pseudotuberculosis virulence and the requirement for the Ysc type III secretion system (T3SS), we utilized two distinct murine models of HH: hemojuvelin knockout mice that mimic severe, early-onset HH as well as mice with the HfeC282Y∕C282Y mutation carried by 10% of people of Northern European descent, associated with adult-onset HH. Hjv−∕− and HfeC282Y∕C282Y transgenic mice displayed enhanced colonization of deep tissues by Y. pseudotuberculosis following oral inoculation, recapitulating enhanced susceptibility of humans with HH to disseminated infection with enteropathogenic Yersinia. Importantly, HH mice orally infected with Y. pseudotuberculosis lacking the T3SS-encoding virulence plasmid, pYV, displayed increased deep tissue colonization relative to wildtype mice. Consistent with previous reports using monocytes from HH vs. healthy donors, macrophages isolated from HfeC282Y∕C282Y mice were defective in Yersinia uptake compared to wildtype macrophages, indicating that the anti-phagocytic property of the Yersinia T3SS plays a less important role in HH animals. These data suggest that Yersinia may rely on distinct virulence factors to cause disease in healthy vs. HH hosts.

Resveratrol mediates therapeutic hepatic effects in acquired and genetic murine models of iron-overload.

Abnormal iron metabolism and hepatic iron-overload is a major cause of liver injury and in the development of chronic liver diseases. Iron-overload-mediated liver disease leads to end-stage cirrhosis and/or hepatocellular carcinoma. Using a genetic hemochromatosis (hemojuvelin knockout mice) and non-genetic (secondary iron-overload) murine models of hepatic iron-overload, we elucidated the mechanism of hepatic iron injury and the therapeutic effects of resveratrol. Hepatic iron-overload was associated with hepatosplenomegaly, increased oxidative stress, hepatic fibrosis, and inflammation, and a pro-apoptotic state which was markedly corrected by resveratrol therapy. Importantly our aging studies with the hemojuvelin knockout mice showed advanced liver disease in association with steatosis in the absence of a diabetic state which recapitulates the essential pathological features seen in clinical iron-overload. Chronic hepatic iron-overload showed increased nuclear localization of acetylated Forkhead fox-O-1 (FoxO1) transcription factor whereas resveratrol dietary intervention reversed the acetylation of FoxO1 in association with increased SIRT1 levels which together with its pleotropic antioxidant properties are likely key mechanisms of its therapeutic action. Importantly, resveratrol treatment did not affect the degree of hepatic iron-overload but rather direct protects the liver from iron-mediated injury. Our findings illustrate a novel and definitive therapeutic action of resveratrol and represent an economically feasible therapeutic intervention to treat hepatic iron-overload and liver disease.

Deficits of learning and memory in Hemojuvelin knockout mice.

Iron is involved in various physiological processes of the human body to maintain normal functions. Abnormal iron accumulation in brain has been reported as a pathogenesis of several neurodegenerative disorders and cognitive impairments. Hemojuvelin (HVJ) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition known as juvenile hemochromatosis. Although iron accumulation in brain has been related to neurodegenerative diseases, it remains unknown the effect of mutation of HVJ gene on cognitive performance. In our studies, HJV(−/−) mice showed deficits in novel object recognition and Morris water maze tests. Furthermore, the expression ration of apoptotic marker Bax and anti-apoptotic marker Bcl-2 in the hippocampus and prefrontal cortex showed higher levels in HJV(−/−) mice. Our results suggested that deletion of HJV gene could increase apoptosis in brain which might contribute to learning and memory deficits in mutant mice. These results indicated that HJV(−/−) mice would be a useful model to study cognitive impairment induced by iron overload in brain.

Retraction: Deficits of learning and memory in Hemojuvelin knockout mice.

This article has been retracted at the request of the authors.

Identification of Differentially Expressed Genes in Mice with Nutritional or Genetic Causes of Iron Overload

Iron overload causes the generation of reactive oxygen species, which can lead to lasting organ damage, particularly to the liver. In patients with hereditary hemochromatosis, transfusion-dependent anemias, and hemoglobinopathies, iron overload is a major cause of mortality. A deeper understanding of iron regulation and the biological pathways involved in maintaining homeostasis may reveal new therapeutic targets for patients with iron overload disorders. We designed this study to discover genes that are differentially expressed in nutritional and genetic models of iron overload. For the nutritional iron overload study, 5-week old male C57BL/6 mice were placed on a soy-free diet (AIN-93G) containing different amounts of iron per kilogram of food: iron-deficient (2.5 mg/kg, n=3), iron-sufficient (37.5 mg/kg, n=3), and iron-excess (750 mg/kg, n=3). In the second study, 5-week old male C57BL/6 mice that were either wild type or HJV knockout mice that exhibited severe early onset iron overload secondary to homozygous deficiency of the bone morphogenic protein coreceptor, hemojuvelin (HJV), were maintained on the iron-deficient (2.5 mg/kg iron) diet (n=2 per group). For both studies animals were sacrificed after 50 days and liver RNA was extracted and sequenced at 40-50 million reads per sample. The RNA integrity number (RIN) for each sample was >6 and assessments of read duplication, base call frequency, and read quality indicated excellent quality of the data. For the HJV knockout mice, we used a false discovery rate <0.05 and a mean-fold change >2, to reveal genes that were differentially expressed compared to wild type mice. For the dietary iron study, genes were grouped by self-organizing maps to identify transcripts whose level of expression trended with increased or decreased dietary iron intake. The resulting analysis identified 148 genes in nutritionally iron-overloaded mice and 688 genes in HJV knockout mice that exhibited significant changes in expression. Of these, 28 genes were differentially regulated in both nutritionally iron overloaded and HJV knockout mice, including expected genes, such as transferrin receptor, HAMP (hepcidin), and bone morphogenic protein 6, and unexpected genes such as cytochrome P450 17a1 (cyp17a1), an enzyme that catalyzes critical steps in steroid synthesis, and nicotinomide N-methyltransferase (nnmt), an enzyme that regulates drug metabolism and DNA methylation. We clustered the 688 differentially expressed genes from the HJV knockout mice into functional pathways using the Functional Analysis tool from DAVID Bioinformatics Resources 6.7 (NIAID). Clusters were considered significant if there were >2 genes in the pathway and the Benjamini-Hochberg P-value was <0.05. We found that the expression of genes involved with PPAR signaling (P=0.0086) was decreased, while expression of transcripts involved with Huntington’s disease (P=0.038) was increased in HJV knockout mice compared to wild-type mice. Our RNA sequencing analysis identified a variety of novel pathways that were differentially regulated in dietary and genetic models of iron overload. Further studies are underway to characterize the potential roles of these genes in iron homeostasis. DisclosuresNo relevant conflicts of interest to declare.

MRNA regulation of cardiac iron transporters and ferritin subunits in a mouse model of iron overload

Iron cardiomyopathy is the leading cause of death in iron overload. Men have twice the mortality rate of women, though the cause is unknown. In hemojuvelin-knockout mice, a model of the disease, males load more cardiac iron than females. We postulated that sex differences in cardiac iron import cause differences in cardiac iron concentration. Reverse transcription polymerase chain reaction was used to measure mRNA of cardiac iron transporters in hemojuvelin-knockout mice. No sex differences were discovered among putative importers of nontransferrin-bound iron (L-type and T-type calcium channels, ZRT/IRT-like protein 14 zinc channels). Transferrin-bound iron transporters were also analyzed; these are controlled by the iron regulatory element/iron regulatory protein (IRE/IRP) system. There was a positive relationship between cardiac iron and ferroportin mRNA in both sexes, but it was significantly steeper in females (p < 0.05). Transferrin receptor 1 and divalent metal transporter 1 were more highly expressed in females than males (p < 0.01 and p < 0.0001, respectively), consistent with their lower cardiac iron levels, as predicted by IRE/IRP regulatory pathways. Light-chain ferritin showed a positive correlation with cardiac iron that was nearly identical in males and females (R(2) = 0.41, p < 0.01; R(2) = 0.56, p < 0.05, respectively), whereas heavy-chain ferritin was constitutively expressed in both sexes. This represents the first report of IRE/IRP regulatory pathways in the heart. Transcriptional regulation of ferroportin was suggested in both sexes, creating a potential mechanism for differential set points for iron export. Constitutive heavy-chain-ferritin expression suggests a logical limit to cardiac iron buffering capacity at levels known to produce heart failure in humans.

OC-15 Accelerated chemically-induced liver fibrosis in hemojuvelin knockout mice mediated by early induction of profibrogenic cytokines

OC-15 Accelerated chemically-induced liver fibrosis in hemojuvelin knockout mice mediated by early induction of profibrogenic cytokines

Interdependence of cardiac iron and calcium in a murine model of iron overload

Iron cardiomyopathy in ß-thalassemia major patients is associated with a vitamin D deficiency. Stores of 25-OH-D3 are markedly reduced, whereas the active metabolite, 1-25-(OH)-D3, is normal or increased. Interestingly, the ratio of 25-OH-D3 to 1-25-(OH)-D3 (a surrogate for parathyroid hormone [PTH]) is the strongest predictor of cardiac iron. Increased PTH and 1-25-OH-D3 levels have been shown to up-regulate L-type voltage-gated calcium channels (LVGCC), the putative channel for cardiac iron uptake. Therefore, we postulate that a vitamin D deficiency increases cardiac iron by altering LVGCC regulation. Hemojuvelin knockout mice were calcitriol treated, PTH treated, vitamin D-depleted, or untreated. Half of the animals in each group received the Ca(2+)-channel blocker verapamil. Mn(2+) was infused to determine LVGCC activity. Hearts and livers were harvested for iron, calcium, and manganese measurements as well as histology. Cardiac iron did not differ among the treatment groups; however, liver iron was increased in vitamin D-depleted animals (P < 0.0003). Cardiac iron levels did not correlate with manganese uptake but were proportional to cardiac calcium levels (r(2) = 0.6; P < 0.0001). Verapamil treatment reduced both cardiac (P < 0.02) and hepatic (P < 0.003) iron levels significantly by 34% and 28%, respectively. The association between cardiac iron and calcium levels was maintained after verapamil treatment (r(2) = 0.3; P < 0.008). Vitamin D depletion is associated with an increase in liver, but not cardiac, iron accumulation. Cardiac iron uptake was strongly correlated with cardiac calcium stores and was significantly attenuated by verapamil, suggesting that cardiac calcium and iron are related.