Bmp6 Expression Can Be Regulated Independently of Liver Iron in Mice

The liver is the primary organ for storing iron and plays a central role in the regulation of body iron levels by secretion of the hormone Hamp1. Although many factors modulate Hamp1 expression, their regulatory mechanisms are poorly understood. Here, we used conditional knockout mice for the iron exporter ferroportin1 (Fpn1) to modulate tissue iron in specific tissues in combination with iron-deficient or iron-rich diets and transferrin (Tf) supplementation to investigate the mechanisms underlying Hamp1 expression. Despite liver iron overload, expression of bone morphogenetic protein 6 (Bmp6), a potent-stimulator of Hamp1 expression that is expressed under iron-loaded conditions, was decreased. We hypothesized that factors other than liver iron must play a role in controlling Bmp6 expression. Our results show that erythropoietin and Tf-bound iron do not underlie the down-regulation of Bmp6 in our mice models. Moreover, Bmp6 was down-regulated under conditions of high iron demand, irrespective of the presence of anemia. We therefore inferred that the signals were driven by high iron demand. Furthermore, we also confirmed previous suggestions that Tf-bound iron regulates Hamp1 expression via Smad1/5/8 phosphorylation without affecting Bmp6 expression, and the effect of Tf-bound iron on Hamp1 regulation appeared before a significant change in Bmp6 expression. Together, these results are consistent with novel mechanisms for regulating Bmp6 and Hamp1 expression.     

Bmp6 Expression in Murine Liver Non Parenchymal Cells: A Mechanism to Control their High Iron Exporter Activity and Protect Hepatocytes from Iron Overload?

Bmp6 is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. Bmp6 expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of Bmp6 in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (Tmprss6 KO mouse) and overload (Hjv KO mouse). With manipulation of dietary iron in wild-type mice, Bmp6 and Tfr1 expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (Hjv KO mice) and deficiency (Tmprss6 KO mice), Bmp6 expression in NPCs was not related to Tfr1. Despite the low Bmp6 in NPCs from Tmprss6 KO mice, Tfr1 mRNA was also low. Conversely, despite body iron overload and high expression of Bmp6 in NPCs from Hjv KO mice, Tfr1 mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that Bmp6 in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production.     

Calcitonin increases hepatic hepcidin expression through the BMP6 of kidney in mice

BackgroundOsteoporosis is frequently accompanied by iron disorders. Calcitonin (CT) was approved as a clinical drug to treat osteoporosis. Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin deficiency leads to iron overload diseases. This study was aimed at investigating the effect of CT on hepatic hepcidin and the mechanism by which CT modulates hepatic hepcidin pathways and iron metabolism.MethodRT-PCR, Western blot, ELISA and siRNA were used to detect the effect of CT on iron metabolism in vivo and in vitro. In addition, the regulatory signal molecules of hepcidin were measured to explore the molecular mechanism of its regulation.ResultsThe results showed that CT strongly increased hepcidin expression and altered iron homeostasis, after mice were intraperitoneal injection of CT. In response to CT administration, BMP6 level in kidney and the serum BMP6 was increased significantly. The phosphorylation of Smad1/5/8 proteins in liver was increased at 3 h and 6 h. Moreover, the Bmp inhibitor LDN-193,189 pretreatment significantly attenuated the CT-mediated increases in phosphorylated Smad1/5/8 and Hamp1 mRNA levels. Calcitonin receptor (CTR) siRNA transfection significant suppressed the role of CT on BMP6 expression in Caki-1 cells.ConclusionOur results suggest that CT strongly induces hepcidin expression and affected iron metabolism. It will provide a new strategy for the treatment of calcium iron related diseases.     

Increased Iron Loading Induces Bmp6 Expression in the Non-Parenchymal Cells of the Liver Independent of the BMP-Signaling Pathway

Bone morphogenetic protein 6 (BMP6) is an essential cytokine for the expression of hepcidin, an iron regulatory hormone secreted predominantly by hepatocytes. Bmp6 expression is upregulated by increased iron-levels in the liver. Both hepatocytes and non-parenchymal liver cells have detectable Bmp6 mRNA. Here we showed that induction of hepcidin expression in hepatocytes by dietary iron is associated with an elevation of Bmp6 mRNA in the non-parenchymal cells of the liver. Consistently, incubation with iron-saturated transferrin induces Bmp6 mRNA expression in isolated hepatic stellate cells, but not in hepatocytes. These observations suggest an important role of the non-parenchymal liver cells in regulating iron-homeostasis by acting as a source of Bmp6.     

Age-related changes in iron homeostasis in mouse ferroxidase mutants

Disorders of iron metabolism are a significant problem primarily in young and old populations. In this study, We compared 1-year-old C57BL6/J mice on iron deficient, iron overload, or iron sufficient diets with two similarly aged genetic models of disturbed iron homeostasis, the sla (sex-linked anemia), and the ceruloplasmin knockout mice (Cp ^−/−) on iron sufficient diet. We found tissue specific changes in sla and nutritional iron deficiency including decreased liver Hamp1 expression and increased protein expression of the enterocyte basolateral iron transport components, hephaestin and ferroportin. In contrast, the Cp ^−/− mice did not show significantly increased Hamp1 expression despite increased liver iron suggesting that regulation is independent of liver iron levels. Together, these results suggest that older mice have a distinct response to alterations in iron metabolism and that age must be considered in future studies of iron metabolism.     

Development of insulin resistance preceded major changes in iron homeostasis in mice fed a high-fat diet

Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) have been associated with dysregulation of iron metabolism. The basis for this association is not completely understood. To attempt to investigate this, we studied temporal associations between onset of insulin resistance (IR) and dysregulated iron homeostasis, in a mouse model of T2DM.Male C57Bl/6 mice (aged 8 weeks) were fed a high-fat diet (HFD; 60% energy from fat) or a control diet (CD; 10% energy from fat) for 4, 8, 12, 16, 20 and 24 weeks. Development of IR was documented, and various metabolic, inflammatory and iron-related parameters were studied in these mice.HFD-feeding induced weight gain, hepato-steatosis and IR in the mice. Onset of IR occurred from 12 weeks onwards. Hepatic iron stores progressively declined from 16 weeks onwards. Accompanying changes included a decrease in hepatic hepcidin (Hamp1) mRNA expression and serum hepcidin levels and an increase in iron content in the epididymal white adipose tissue (eWAT). Iron content in the liver negatively correlated with that in the eWAT. Factors known to regulate hepatic Hamp1 expression (such as serum iron levels, systemic inflammation, and bone marrow-derived erythroid regulators) were not affected by HFD-feeding. In conclusion, the results show that the onset of IR in HFD-fed mice preceded dysregulation of iron homeostasis, evidence of which were found both in the liver and visceral adipose tissue.     

Iron loading and oxidative stress in the Atm-/- mouse liver

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ～30% of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated (Atm) null mice on control or high-iron diets. Atm(-/-) mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm(-/-) mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased; however, protein levels were unchanged. We demonstrate that Atm(-/-) mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.     

Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity.

Using a murine model, we studied the effect of agonistic anti-CD95 antibodies (aCD95) on sinusoidal lining cells and a potential protection by caspase inhibition. C3H/HeN mice were intravenously administered aCD95 (10 microgram/mouse) or unspecific IgG (control) in the presence or absence of the caspase inhibitor z-VAD-fmk. Analysis of hepatic microcirculation using intravital fluorescence microscopy revealed severe (P<0.01) sinusoidal perfusion failure and reduced (P<0.05) phagocytic activity of Kupffer cells (KC) within 2 h. Transmission electron micrographs demonstrated loss of integrity of sinusoidal endothelial cells as early as 1 h after aCD95 application, whereas histological manifestation of hepatocellular apoptosis and hemorrhagic necrosis was most pronounced at 6 h. Blocking of caspase activity attenuated (P<0.01) both hepatic microvascular perfusion failure and KC dysfunction. Accordingly, full protection of the liver from apoptotic damage and intact microarchitecture was observed in histological sections after z-VAD-fmk treatment. Mortality rate was 40% 6 h after aCD95 administration, whereas all animals survived in the z-VAD-fmk group (P<0.05). The activation of caspases through CD95 may primarily lead to damage of sinusoidal endothelial cells and hepatic microvascular perfusion failure. Moreover, reduced phagocytic capacity of KC may contribute to accumulation of toxic metabolites released by dying cells at the local site of inflammation, further aggravating liver injury.     

Ultrastructural observations in the carbonyl iron-fed rat, an animal model for hemochromatosis

Rats fed a carbonyl iron-supplemented diet for 4-15 months were studied for iron content and morphologic changes in the liver, spleen, intestinal mucosa, pancreas and heart. All organs had an increased iron content measured by atomic absorption, with the highest concentrations in the liver and spleen. The periportal distribution of stored iron in the liver was similar to that in human hemochromatosis. In animals treated beyond 6 months Kupffer cells and sinusoidal lining cells also showed cytosiderosis. Electron microscopy provided information on ferritin and hemosiderin content and distribution within parenchymal and sinusoidal cells of the liver but no excessive fibrosis was found. Except for the spleen, the other organs showed less iron deposition. Iron-filled lysosomes (siderosomes) were found in macrophages in the intestinal lamina propria and pancreas, as well as in enterocytes, pancreatic acinar cells and heart muscle cells. Heavily iron-laden siderosomes had increased membrane instability which was demonstrated both morphologically and by measurements of latent lysosomal enzyme activities. Even though cirrhosis was not found, the distribution pattern of accumulated storage iron and lysosomal lability indicated that the carbonyl iron-fed rat is a suitable experimental model for human hemochromatosis.     

Vitamin A deficiency modulates iron metabolism via ineffective erythropoiesis

Vitamin A modulates inflammatory status, iron metabolism and erythropoiesis. Given that these factors modulate the expression of the hormone hepcidin (Hamp), we investigated the effect of vitamin A deficiency on molecular biomarkers of iron metabolism, the inflammatory response and the erythropoietic system. Five groups of male Wistar rats were treated: control (AIN-93G), the vitamin A-deficient (VAD) diet, the iron-deficient (FeD) diet, the vitamin A- and iron-deficient (VAFeD) diet or the diet with 12 mg atRA/kg diet replacing all-trans-retinyl palmitate by all-trans retinoic acid (atRA). Vitamin A deficiency reduced serum iron and transferrin saturation levels, increased spleen iron concentrations, reduced hepatic Hamp and kidney erythropoietin messenger RNA (mRNA) levels and up-regulated hepatic and spleen heme oxygenase-1 gene expression while reducing the liver HO-1 specific activity compared with the control. The FeD and VAFeD rats exhibited lower levels of serum iron and transferrin saturation, lower iron concentrations in tissues and lower hepatic Hamp mRNA levels compared with the control. The treatment with atRA resulted in lower serum iron and transferrin concentrations, an increased iron concentration in the liver, a decreased iron concentration in the spleen and in the gut, and decreased hepatic Hamp mRNA levels. In summary, these findings suggest that vitamin A deficiency leads to ineffective erythropoiesis by the down-regulation of renal erythropoietin expression in the kidney, resulting in erythrocyte malformation and the consequent accumulation of the heme group in the spleen. Vitamin A deficiency indirectly modulates systemic iron homeostasis by enhancing erythrophagocytosis of undifferentiated erythrocytes.     

Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate

The cellular uptake and storage of iron have to be tightly regulated in order to provide iron for essential cellular functions while preventing the iron-catalysed generation of reactive oxygen species (ROS). In contrast to cells in other organs, little is known about the regulation of iron metabolism in brain cells, particularly in astrocytes. To investigate the regulation of iron metabolism in astrocytes we have used primary astrocyte cultures from the brains of newborn rats. After application of ferric ammonium citrate (FAC), cultured astrocytes accumulated iron in a time- (0-48 h) and concentration-dependent (0.01-1 mm) manner. This accumulation was prevented if FAC was applied in combination with the iron-chelator deferoxamine (DFX). Application of FAC to astrocyte cultures caused a strong increase in the cellular content of the iron storage protein ferritin and a decrease in the amount of transferrin receptor (TfR), which is involved in the transferrin-mediated uptake of iron into cells. In contrast, application of DFX strongly increased the level of TfR. Both up-regulation of ferritin content by iron application and up-regulation of TfR content by DFX were prevented by the protein synthesis inhibitor cycloheximide (CHX). During incubation of astrocytes with FAC, a mild and transient increase in the extracellular activity of the cytosolic enzyme lactate dehydrogenase and in the concentration of intracellular ROS was observed. In contrast, prevention of protein synthesis by CHX during incubation with FAC resulted in significantly more cell loss and a persistent and intense increase in the production of intracellular ROS. These results demonstrate that both iron accumulation and deprivation modulate the synthesis of ferritin and TfR in astrocytes and that protein synthesis is required to prevent iron-mediated toxicity in astrocytes.     

Enhanced insulin signaling and its downstream effects in iron-overloaded primary hepatocytes from hepcidin knock-out mice

BackgroundIncreased body iron stores have been implicated in the pathogenesis of diabetes mellitus. However, the molecular mechanisms involved are unclear. The liver plays a central role in homeostasis of iron and glucose in the body. Mice deficient in hepcidin (the central regulator of systemic iron homeostasis) (Hamp1^−/− mice) accumulate iron in the liver in vivo. The effects of such iron loading on hepatic insulin signaling and glucose metabolism are not known.MethodsHepatocytes isolated from Hamp1^−/− mice were studied for markers of insulin signaling (and its downstream effects), glucose production, expression of gluconeogenic and lipogenic enzymes, and markers of AMPK (AMP-activated protein kinase) activation and oxidative stress. These parameters were studied both in the absence and presence of insulin, and also with the use of an iron chelator.ResultsAkt in the insulin signaling pathway was found to be activated in the Hamp1^−/− hepatocytes to a greater extent than wild-type (WT) cells, both under basal conditions and in response to insulin. Incubation of the Hamp1^−/− hepatocytes with an iron chelator attenuated these effects. There was no evidence of oxidative stress or AMPK activation in the Hamp1^−/− hepatocytes. Glucose production by these cells was similar to that by WT cells. Gene expression of key gluconeogenic enzymes was decreased in these cells. In addition, they showed evidence of increased lipogenesis.ConclusionsHepatocytes from Hamp1^−/− mice showed evidence of greater sensitivity to the effects of insulin than WT hepatocytes. This may explain the insulin-sensitive phenotype that has been reported in classical hemochromatosis.     

Bmp and Fgf signaling are essential for liver specification in zebrafish

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.     

Iron prevents ferritin turnover in hepatic cells

It has long been assumed that iron regulates the turnover of ferritin, but evidence for or against this idea has been lacking. This issue was addressed using rat hepatoma cells with characteristics of hepatocytes subjected to a continuous influx of iron. Iron-pretreated cells were pulsed with [(35)S]Met for 60 min or with (59)Fe overnight and harvested up to 30 h thereafter, during which they were/were not cultured with ferric ammonium citrate (FAC; 180 microm). Radioactivity in ferritin/ferritin subunits of cell heat supernatants was determined by autoradiography of rockets obtained by immunoelectrophoresis or after precipitation with ferritin antibody and SDS-PAGE. Both methods gave similar results. During the +FAC chase, the concentration of ferritin in the cells increased linearly with time. Without FAC, the half-life of (35)S-ferritin was 19-20 h; with FAC there was no turnover. Without FAC, the iron in ferritin had an apparent half-life of 20 h; in the presence of FAC there was no loss of (59)Fe. Without FAC, concentrations of ferritin iron and protein also decreased in parallel. We conclude that a continuous influx of excess iron can completely inhibit the degradation of ferritin protein and that the iron and protein portions of ferritin molecules may be coordinately degraded.     

Smad6 and Smad7 are co-regulated with hepcidin in mouse models of iron overload

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe ?/? and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.