Iron Homeostasis In Response Research Articles

Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E.coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E.coli Fur also binds a [2Fe-2S] cluster in wildtype E.coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box invitro, and are inactive to complement the function of Fur invivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E.coli cells.

Ferric Uptake Regulator (Fur) Regulates Intracellular Iron Homeostasis via reversible binding of a [2Fe‐2S] cluster in Escherichia coli

It has generally been assumed that when intracellular “free” iron content is elevated in bacteria, a global transcription factor Fur (Ferric uptake regulator) binds Fe(II) to regulate expression of its target genes and achieve intracellular iron homeostasis. Fur is highly conserved among bacteria, and purified Fur proteins from different bacteria may exist as a homodimer or tetramer with each monomer containing three metal binding sites. While two of the metal binding sites (site 1 and site 2) in purified Fur proteins are often occupied by Zn(II), the iron‐bound Fur has never been isolated from any bacteria. Here we report that Escherichia coli Fur binds a [2Fe‐2S] cluster at site 3 (via Cys‐93 and Cys‐96) in the E. coli mutant cells in which intracellular “free” iron content is elevated due to deficiency of iron‐sulfur cluster biogenesis. Depletion of intracellular “free” iron content effectively removes the [2Fe‐2S] cluster from Fur, indicating that binding of a [2Fe‐2S] cluster in Fur is determined by intracellular “free” iron content. Fur homolog from Haemophilus influenzae also binds a [2Fe‐2S] cluster at site 3 when expressed in E. coli cells, suggesting that binding of a [2Fe‐2S] cluster in Fur is highly conserved. While the Zn(II)‐bound E. coli Fur has a basal binding activity for its target DNA sequence known as Fur‐box, binding of a [2Fe‐2S] cluster significantly enhances its Fur‐box binding activity. Similarly, binding of a [2Fe‐2S] cluster is required for the strong binding activity of H. influenzaeFur for Fur‐box in vitro. Mutation of Cys‐93 to Ala results in an E. coli Fur mutant that fails to bind the [2Fe‐2S] cluster, loses the enhanced binding activity for Fur‐box, and is inactive to complement the physiological function of E. coli Fur in E. coli cells. Our results suggest that E. coli Fur and H. influenzae Fur binds a [2Fe‐2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular “free” iron content in cells.

Regulation of macrophage iron homeostasis is associated with the localization of bacteria.

Iron not only plays an important role in the physiological function of organisms but also is an essential nutrient for the growth of pathogens. There is a competing relationship between organisms and pathogens for the use of iron in the case of infection. The macrophage, as the first immune cell found to participate in iron metabolism, has a precise regulation system to maintain iron homeostasis in response to pathogen infection. However, few studies have compared the effects of different types of bacterial infections on the iron homeostasis of macrophages. In this study, we investigated the changes in the iron regulation of the macrophage 3D4/2 by the infection of the extracellular bacterium Escherichia coli K88 (E. coli K88) and the intracellular bacterium Salmonella typhimurium (S. typhimurium). We found that S. typhimurium infection reduced the uptake of extracellular iron, promoted the outflow transport of intracellular iron, and decreased the free iron ions for intracellular bacterial proliferation and utilization. However, the infection of E. coli K88 reversed iron regulation by promoting the uptake of extracellular iron, reducing the extracellular transport of intracellular iron and increasing the storage of iron in 3D4/2. The results demonstrated that macrophages had completely opposing regulations of iron metabolism in response to intracellular and extracellular bacteria. It suggested that the diversion of cellular iron traffic would be considered as an important defense mechanism for macrophages to reduce iron availability for bacteria, and the resistance of iron spread or the interruption of the assimilation of iron by bacteria would be beneficial in developing therapeutics for bacterial infection.

Advances in iron homeostasis and ferromagnetic nanoparticles

The essential trace element iron is a major structural component required for the synthesis and function of hemoglobin and various enzymes, and is therefore involved in many key physiological processes, including oxygen transport, nucleic acid synthesis, gene expression, and immune regulation. Iron homeostasis is therefore essential to maintaining health. For example, iron deficiency leads to anemia, whereas iron overload can lead to the formation of free radicals, which cause lipid peroxidation, DNA damage, and changes in other bioactive molecules. Thus, iron overload has been implicated in a wide range of diseases, including hemochromatosis, diabetes, cardiovascular disease, neurodegeneration, and cancer. Systemically, iron homeostasis is tightly regulated by several processes, including iron absorption in the intestine, iron circulation in the blood, iron utilization in various tissues, iron storage in the liver, and iron recycling in splenic red pulp macrophages. In recent years, new genetic animal models and cutting-edge technologies have led to considerable progress regarding the molecular mechanisms that underlie iron metabolism. Ferroptosis, a recently identified iron-dependent form of cell death, has been implicated in several disease conditions, including acute kidney failure following ischemia/reperfusion, Huntingtons disease, cancer, and neurodegeneration. Although no definitive biomarkers have been identified, lipid peroxidation, PTGS2 expression, and NADPH levels have been suggested as possible biomarkers of ferroptosis. Nevertheless, the regulatory mechanisms underlying ferroptosis are fairly complex and poorly understood. Recently, our understanding of the pathways that regulate hepcidin, the master regulator of iron homeostasis, has increased considerably. Hepcidin regulates systemic iron levels by facilitating degradation of the sole iron exporter, Ferroportin, thereby controlling the absorption, utilization, and storage of iron. Hepcidin also cooperatively regulates iron homeostasis in response to various stimuli; specifically, iron overload and inflammation upregulate hepcidin expression, whereas erythropoiesis and hypoxic stress downregulate hepcidin expression. In addition, several epigenetic processes regulate hepcidin expression, thereby regulating iron metabolism. New studies are continually revealing novel molecular mechanisms related to the complex networks that regulate hepcidin, providing exciting new possibilities for treating iron-related diseases. Iron-containing biological materials have also been studied for their clinical potential. For example, ferromagnetic nanoparticles, which are ferritin- or Fe3O4-containing biomacromolecules, have been applied in a wide variety of biomedical fields due to their superior magnetic properties and biocompatibility, making them ideal drug carriers. Importantly, nanoparticles readily penetrate the blood-brain barrier and enter the central nervous system, improving the bioavailability and therapeutic efficacy of drugs. Alternatively, an external magnetic field can be used to direct ferromagnetic nanoparticles to specific lesions. For example, a drug-containing transferrin nanoparticle complex could be used to targets specific tumor cells due to their increased expression of the transferrin receptor compared to healthy tissue. Superparamagnetic iron oxide nanoparticles may also be a promising tool for destroying cancer cells using a rapidly alternating external magnetic field. Thanks to their wide range of properties and features, ferromagnetic nanoparticles offer a wealth of promising biomedical applications. Here, we review the molecular mechanisms that regulate iron homeostasis, focusing on groundbreaking discoveries in hepcidin regulation and recent progress regarding biomedical applications for ferromagnetic nanoparticles.

Candida albicans Hap43 Domains Are Required under Iron Starvation but Not Excess.

Iron availability is a central factor in infections, since iron is a critical micronutrient for all living organisms. The host employs both iron limitation and toxicity strategies to control microbial growth, and successful pathogens are able to tightly coordinate iron homeostasis in response to changing iron levels. As a commensal and opportunistic pathogen, Candida albicans copes with both iron deficiency and excess via the precise regulation of iron acquisition, consumption and storage. The C. albicans transcription factor Hap43 is known to be required for the iron starvation response, while specific domains of its ortholog, HapX, in Aspergillus fumigatus, were recently shown to regulate iron uptake and consumptions genes under both low and high iron levels. Therefore, we investigated the contribution of C. albicans Hap43 domains in response to changing iron levels. We found the C-terminus of Hap43 to be essential for the activation of iron uptake genes during iron starvation, whereas, in contrast to A. fumigatus, Hap43 was not required in mediating adaptation to iron resistance. These data indicate that the generally conserved metal acquisition systems in fungal pathogens can show individual adaptations to the host environment.

Redox Sensing by Fe2+ in Bacterial Fur Family Metalloregulators.

Iron is required for growth and is often redox active under cytosolic conditions. As a result of its facile redox chemistry, iron homeostasis is intricately involved with oxidative stress. Bacterial adaptation to iron limitation and oxidative stress often involves ferric uptake regulator (Fur) proteins: a diverse set of divalent cation-dependent, DNA-binding proteins that vary widely in both metal selectivity and sensitivity to metal-catalyzed oxidation. Recent Advances: Bacteria contain two Fur family metalloregulators that use ferrous iron (Fe2+) as their cofactor, Fur and PerR. Fur functions to regulate iron homeostasis in response to changes in intracellular levels of Fe2+. PerR also binds Fe2+, which enables metal-catalyzed protein oxidation as a mechanism for sensing hydrogen peroxide (H2O2). To effectively regulate iron homeostasis, Fur has an Fe2+ affinity tuned to monitor the labile iron pool of the cell and may be under selective pressure to minimize iron oxidation, which would otherwise lead to an inappropriate increase in iron uptake under oxidative stress conditions. Conversely, Fe2+ is bound more tightly to PerR but exhibits high H2O2 reactivity, which enables a rapid induction of peroxide stress genes. The features that determine the disparate reactivity of these proteins with oxidants are still poorly understood. A controlled, comparative analysis of the affinities of Fur/PerR proteins for their metal cofactors and their rate of reactivity with H2O2, combined with structure/function analyses, will be needed to define the molecular mechanisms that have facilitated this divergence of function between these two paralogous regulators.

The protective role of TET2 in erythroid iron homeostasis against oxidative stress and erythropoiesis

Although previous studies suggested that stress erythropoiesis and iron metabolism regulate each other to increase iron availability for hemoglobin synthesis, the molecular bases determining its different traits remain elusive. In addition, global DNA demethylation has been reported during mouse erythropoiesis in vivo. However, the understanding of iron-related genes through DNA demethylation under stress erythropoiesis is largely unknown. In the current study, we found disordered iron homeostasis and misregulated hepcidin-ferroportin axis under stress erythropoiesis. Interestingly, global 5hmC content and TET2 expression were significantly induced by oxidative stress, whereas antioxidant had the opposite's effect. Mechanistic investigation manifested that TET2-mediated DNA demethylation promotes the expression of ferroportin and erythroferrone against oxidative stress. Besides, the expression of NRF2 was significantly increased by TET2-mediated DNA demethylation during stress erythropoiesis. Elevated NRF2 expression could also modulate the activation of ferroportin and erythroferrone through a canonical antioxidant response element within its promoter. These direct and indirect pathways of TET2 synergistically cooperated to mediating iron metabolism during stress erythropoiesis. Our work revealed a critical role of TET2-mediated DNA demethylation against oxidative stress, and provided the molecular mechanisms underlying the epigenetic regulation of iron homeostasis in response to stress erythropoiesis.

Modelling Systemic Iron Regulation during Dietary Iron Overload and Acute Inflammation: Role of Hepcidin-Independent Mechanisms.

Systemic iron levels must be maintained in physiological concentrations to prevent diseases associated with iron deficiency or iron overload. A key role in this process plays ferroportin, the only known mammalian transmembrane iron exporter, which releases iron from duodenal enterocytes, hepatocytes, or iron-recycling macrophages into the blood stream. Ferroportin expression is tightly controlled by transcriptional and post-transcriptional mechanisms in response to hypoxia, iron deficiency, heme iron and inflammatory cues by cell-autonomous and systemic mechanisms. At the systemic level, the iron-regulatory hormone hepcidin is released from the liver in response to these cues, binds to ferroportin and triggers its degradation. The relative importance of individual ferroportin control mechanisms and their interplay at the systemic level is incompletely understood. Here, we built a mathematical model of systemic iron regulation. It incorporates the dynamics of organ iron pools as well as regulation by the hepcidin/ferroportin system. We calibrated and validated the model with time-resolved measurements of iron responses in mice challenged with dietary iron overload and/or inflammation. The model demonstrates that inflammation mainly reduces the amount of iron in the blood stream by reducing intracellular ferroportin transcription, and not by hepcidin-dependent ferroportin protein destabilization. In contrast, ferroportin regulation by hepcidin is the predominant mechanism of iron homeostasis in response to changing iron diets for a big range of dietary iron contents. The model further reveals that additional homeostasis mechanisms must be taken into account at very high dietary iron levels, including the saturation of intestinal uptake of nutritional iron and the uptake of circulating, non-transferrin-bound iron, into liver. Taken together, our model quantitatively describes systemic iron metabolism and generated experimentally testable predictions for additional ferroportin-independent homeostasis mechanisms.

Co-regulation of Iron Metabolism and Virulence Associated Functions by Iron and XibR, a Novel Iron Binding Transcription Factor, in the Plant Pathogen Xanthomonas.

Abilities of bacterial pathogens to adapt to the iron limitation present in hosts is critical to their virulence. Bacterial pathogens have evolved diverse strategies to coordinately regulate iron metabolism and virulence associated functions to maintain iron homeostasis in response to changing iron availability in the environment. In many bacteria the ferric uptake regulator (Fur) functions as transcription factor that utilize ferrous form of iron as cofactor to regulate transcription of iron metabolism and many cellular functions. However, mechanisms of fine-tuning and coordinated regulation of virulence associated function beyond iron and Fur-Fe2+ remain undefined. In this study, we show that a novel transcriptional regulator XibR (named X anthomonas iron binding regulator) of the NtrC family, is required for fine-tuning and co-coordinately regulating the expression of several iron regulated genes and virulence associated functions in phytopathogen Xanthomonas campestris pv. campestris (Xcc). Genome wide expression analysis of iron-starvation stimulon and XibR regulon, GUS assays, genetic and functional studies of xibR mutant revealed that XibR positively regulates functions involved in iron storage and uptake, chemotaxis, motility and negatively regulates siderophore production, in response to iron. Furthermore, chromatin immunoprecipitation followed by quantitative real-time PCR indicated that iron promoted binding of the XibR to the upstream regulatory sequence of operon’s involved in chemotaxis and motility. Circular dichroism spectroscopy showed that purified XibR bound ferric form of iron. Electrophoretic mobility shift assay revealed that iron positively affected the binding of XibR to the upstream regulatory sequences of the target virulence genes, an effect that was reversed by ferric iron chelator deferoxamine. Taken together, these data revealed that how XibR coordinately regulates virulence associated and iron metabolism functions in Xanthomonads in response to iron availability. Our results provide insight of the complex regulatory mechanism of fine-tuning of virulence associated functions with iron availability in this important group of phytopathogen.

Contrasting regulation of macrophage iron homeostasis in response to infection with Listeria monocytogenes depending on localization of bacteria.

Due to its multiple roles for the proliferation and pathogenicity of many microbes on the one hand and via modulation of immune effector functions on the other hand the control over iron homeostasis is thought to play a decisive role in the course of infections. Diversion of cellular iron traffic is considered as an important defense mechanism of macrophages to reduce metal availability for intracellular bacteria residing in the phagosome. However, evidence is lacking whether such alterations of iron homeostasis also become evident upon infection with bacteria gaining access to the cytosol like Listeria monocytogenes. Here we show that infection of macrophages with L. monocytogenes triggers the expression of the major cellular iron exporter ferroportin1 and induces cellular iron egress. As the growth of Listeria within macrophages is promoted by iron, stimulation of ferroportin1 functionality limits the availability of the metal for Listeria residing in the cytoplasm, whereas ferroportin1 degradation upon hepcidin treatment increases intracellular bacterial growth. In parallel to an increase of ferroportin1 expression, infected macrophages induce anti-microbial immune effector mechanisms such as TNFα formation or NO expression which are aggravated upon iron deficiency. These adaptive changes of iron homeostasis and immune response pathways are only found in macrophages infected with Listeria which express listeriolysin O and are therefore able to escape from the phagosome to the cytoplasm. Listeriolysin O deficient Listeria which are restricted to the phagosome are even killed by excess iron which may be based on "iron intoxification" via macrophage radical formation, because iron supplementation in that setting is paralleled by increased ROS formation. Our results indicate that ferroportin1 mediated iron export is a nutritional immune effector pathway to control infection with Listeria residing in the cytoplasm, whereas a different strategy is observed in mutant Listeria restricted to the phagosome, where iron remains in the macrophages likewise contributing to ROS mediated intoxification of bacteria.

PGC-1α Regulates Hepatic Hepcidin Expression and Iron Homeostasis in Response to inflammation

Systemic iron homeostasis is finely regulated by the liver through synthesis of the peptide hormone hepcidin (HAMP), which plays an important role in duodenal iron absorption and macrophage iron release. Clinical investigations have shown that chronic and low-grade inflammation leads to the increase of serum HAMP levels and the development of various diseases such as anemia of inflammation. However, gaps remain to fully elucidate the mechanism linking inflammation and iron dysregulation. Here we show that although inflammatory stimuli increase hepatic HAMP expression and cause systemic iron deficiency in mice, they inhibit the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a transcriptional coactivator actively involved in metabolic regulation. Liver-specific overexpression of PGC-1α antagonizes lipopolysaccharide-induced HAMP expression and alleviates various pathophysiological changes similar to anemia of inflammation. Consistently, overexpression of PGC-1α in HepG2 or HuH7 cells also suppresses HAMP expression and reduces iron accumulation. In contrast, knockdown of PGC-1α exaggerates LPS-induced HAMP expression and iron dysregulation. At the molecular level, PGC-1α suppresses HAMP transcription via the interaction with hepatocyte nuclear factor 4α. In addition, PGC-1α is present near hepatocyte nuclear factor 4α-binding site on the proximal HAMP promoter and turns the chromatin structure into an inactive state. Our data suggest a critical role for PGC-1α in the regulation of hepatic HAMP expression and iron homeostasis under inflammatory circumstances.

The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

Regulation of iron metabolism in Hamp −/− mice in response to iron-deficient diet

Hepcidin, the liver-secreted iron regulatory peptide, maintains systemic iron homeostasis in response to several stimuli including dietary iron levels and body iron status. In addition, iron metabolism is controlled by several local regulatory mechanisms including IRP and Hif-2α activities independently of hepcidin. However, the roles of these mechanisms and their interaction particularly in hepcidin-deficient individuals are not yet fully understood. We, therefore, aimed to explore whether Hamp disruption affects iron homeostatic responses to dietary iron deficiency. Hepcidin1 knockout (Hamp (-/-)) mice and heterozygous littermates were fed with control or iron-deficient diet for 2 weeks. The expression of iron-related genes and proteins were determined by quantitative PCR and Western blot, respectively. Two-week iron-deficient diet feeding in Hamp (-/-) mice did not alter serum iron but significantly reduced liver non-heme iron levels. This was also associated with increased ferroportin protein expression in the duodenum and spleen, whereas decreased expression was found in the liver. In addition, significant inductive effects of iron-deficient diet on Dcytb and DMT1 mRNA expression in the duodenum were noted with more pronounced effects in Hamp (-/-) mice compared with controls. Hamp (-/-) mice exhibited a more dramatic increase in the expression of iron transport machinery, which may be responsible for the unaltered serum iron levels upon iron-deficient diet feeding in these mice. Despite the lack of hepcidin, Hamp (-/-) mice can maintain a degree of iron homeostasis in response to altered dietary iron through several hepcidin-independent mechanisms.

Changes of hepatic lactoferrin gene expression in two mouse models of the acute phase reaction

Lactoferrin (Ltf), an iron binding glycoprotein, is a pleiotropic molecule whose serum concentration increases under acute phase conditions. The physiological roles of this protein have been well elucidated, but the source and serum regulation of Ltf gene expression have not been investigated in detail as part of the acute phase reaction (APR). In the current work, the changes in hepatic Ltf-gene-expression during turpentine oil- (TO-) or LPS-induced APR were investigated. Ltf was upregulated at both the mRNA and protein levels in the liver of TO- and LPS-treated wild type (WT) mice. The pattern of induction however was different in both animal models indicating distinctive signalling patterns resulting in an acute phase reaction. Cytokines are the core regulators of APR. Among the major cytokines, IL-6 is an important signalling molecule, which also regulates iron homeostasis in response to an inflammatory situation. In this study, the administration of IL-6 induced Ltf gene expression in the liver of WT mice, in murine hepatocytes and in hepa 1-6 cells. Ltf-gene-expression was upregulated also in the liver of TO- and LPS-treated IL-6 knockout (KO) mice. The increase in serum Ltf after LPS injection was greater than after TO-injection both in WT and IL-6-KO mice. To evaluate the contribution of other acute phase cytokines in the regulation of Ltf-gene-expression in the liver, both in vitro and in vivo studies with IL-1β, TNF-α, or IFN-γ were performed. The results demonstrate that TNF-α and IFN-γ also upregulated Ltf-gene-expression, while IL-1β has no role in the regulation of Ltf-gene-expression.

Iron Regulatory Protein 1 Outcompetes Iron Regulatory Protein 2 in Regulating Cellular Iron Homeostasis in Response to Nitric Oxide

In mammals, iron regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of genes involved in iron metabolism, including transferrin receptor 1, the ferritin (Ft) H and L subunits, and ferroportin by binding mRNA motifs called iron responsive elements (IREs). IRP1 is a bifunctional protein that mostly exists in a non-IRE-binding, [4Fe-4S] cluster aconitase form, whereas IRP2, which does not assemble an Fe-S cluster, spontaneously binds IREs. Although both IRPs fulfill a trans-regulatory function, only mice lacking IRP2 misregulate iron metabolism. NO stimulates the IRE-binding activity of IRP1 by targeting its Fe-S cluster. IRP2 has also been reported to sense NO, but the intrinsic function of IRP1 and IRP2 in NO-mediated regulation of cellular iron metabolism is controversial. In this study, we exposed bone marrow macrophages from Irp1(-/-) and Irp2(-/-) mice to NO and showed that the generated apo-IRP1 was entirely responsible for the posttranscriptional regulation of transferrin receptor 1, H-Ft, L-Ft, and ferroportin. The powerful action of NO on IRP1 also remedies the defects of iron storage found in IRP2-null bone marrow macrophages by efficiently reducing Ft overexpression. We also found that NO-dependent IRP1 activation, resulting in increased iron uptake and reduced iron sequestration and export, maintains enough intracellular iron to fuel the Fe-S cluster biosynthetic pathway for efficient restoration of the citric acid cycle aconitase in mitochondria. Thus, IRP1 is the dominant sensor and transducer of NO for posttranscriptional regulation of iron metabolism and participates in Fe-S cluster repair after exposure to NO.

Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H(2)O(2), hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

Pathways for the regulation of body iron homeostasis in response to experimental iron overload

Secondary iron overload is a frequent clinical condition found in association with multiple blood transfusions. To gain insight into adaptive changes in the expression of iron genes in duodenum, liver and spleen upon experimental iron overload we studied C57BL/6 mice receiving repetitive daily injections of iron-dextran for up to 5 days. Iron initially accumulated in spleen macrophages but with subsequent increase in macrophage ferroportin and ferritin expression its content in the spleen decreased while a progressive storage of iron occurred within hepatocytes which was paralleled by a significant increase in hepcidin and hemojuvelin expression. Under these conditions, iron was still absorbed from the duodenal lumen as divalent metal transporter-1 expressions were high, however, most of the absorbed iron was incorporated into duodenal ferritin, while ferroportin expression drastically decreased and iron transfer to the circulation was reduced. Experimental iron overload results in iron accumulation in macrophages and later in hepatocytes. In parallel, the transfer of iron from the gut to the circulation is diminished which may be referred to interference of hepcidin with ferroportin mediated iron export, thus preventing body iron accumulation.

Iron Regulatory Proteins, Iron Responsive Elements and Iron Homeostasis

The discovery of iron regulatory proteins (IRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of vertebrate iron metabolism. IRPs bind to iron responsive elements (IREs) in specific mRNAs and regulate their utilization. The targets of IRP action now appear to extend beyond proteins that function in the storage (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic acid cycle. To date, it appears that IRPs modulate the utilization of six mammalian mRNAs. Current studies are aimed at defining the mechanisms responsible for the hierarchical regulation of these mRNAs by IRPs. In addition, much interest continues to focus on the signaling pathways through which IRP function is regulated. Multiple factors modulate the RNA binding activity of IRP1 and/or IRP2 including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress and hypoxia/reoxygenation. Because IRPs are key modulators of the uptake and metabolic fate of iron in cells, they are focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances.