The relation between Lead (Pb) and iron (Fe) becomes increasingly concerned because they are both divalent metals that are absorbed by the same intestinal mechanism, and Pb exposure and Fe deficiency in the developmental brain, as well as Fe overload in the aged brain, can cause cognitive deficits. However, the interaction between Pb exposure and Fe status in the brain has not been established. Therefore, in the current study, we examined the effects of maternal ingestion of Pb in drinking water during gestation and lactation on the Fe status and the expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FP1) in the brain of offspring. The offspring were followed through old age, with measurements taken at postnatal week 3, 41 and 70. Pb exposure increases the Fe content in the old-aged rats' brain, Which might be not subjected to DMT1 mediating, but may be associated with the decrease expression of FP1. Furthermore, the effect of Pb on FP1 expression is regulated at transcriptional and posttranscriptional levels. The perturbation in Fe homeostasis may contribute to the neurotoxicology consequences induced by Pb exposure, and FP1 may play a role in Pb-induced Fe cumulation in the brain.
Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally relevant signal for hepcidin-induced ferroportin endocytosis.
BACKGROUND: Growth differentiation factor 15 (GDF15), a divergent TGFβ superfamily, has recently been implicated in the modulation of iron homeostasis, acting as an upstream negative regulator of hepcidin, the key iron regulatory hormone produced primarily by hepatocytes. However, little is known about possible roles that GDF15 might play in the regulation of iron homeostasis and development of hyperferritinemia in children with hemophagocytic lymphohistiocytosis (HLH). PROCEDURES: We compared serum GDF15 level and mRNA expressions of GDF15 and key molecules of iron metabolism, and made correlations between their expressions in children with HLH and control children. RESULTS: Serum GDF15 level was remarkably higher in HLH group than that in controls, with median serum concentration of 1,700 and 260 pg/ml, respectively (P < 0.001). In addition, GDF15 mRNA was significantly upregulated but independent of hypoxia-inducible factor-mediated oxygen signaling pathway. More importantly, GDF15 induction was positively correlated to upregulation of ferroportin, the only cellular iron exporter, and to upregulation of ferritin heavy chain. CONCLUSIONS: Our study suggests that GDF15 induction helps suppress further activation of macrophages in stressful physiologic states as HLH, and is intimately implicated in the development of hyperferritinemia by modulating the hepcidin-ferroportin axis, resulting in enhanced ferroportin-mediated iron efflux. Pediatr Blood Cancer © 2013 Wiley Periodicals, Inc.
Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron-hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptor (TLR) 2 and TLR6 and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin mRNA and protein expression in bone marrow-derived macrophages, liver and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin down regulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin down regulation in the major sites of iron recycling may represent a first line response to restrict iron access for numerous pathogens.
SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2-related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2-/- mice had decreased iron levels, while this effect was reversed in Sirt2-/- Nrf2-/- double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.
The control over iron homeostasis is critical in host-pathogen-interaction. Iron plays not only multiple roles for bacterial growth and pathogenicity, but also for modulation of innate immune responses. Hepcidin is a key regulator of host iron metabolism triggering degradation of the iron exporter ferroportin. Although iron overload in humans is known to increase susceptibility to Burkholderia pseudomallei, it is unclear how the pathogen competes with the host for the metal during infection. This study aimed to investigate whether B. pseudomallei, the causative agent of melioidosis, modulates iron balance and how regulation of host cell iron content affects intracellular bacterial proliferation.
Hepcidin is a small cysteine rich peptide that regulates the sole known cellular iron exporter, ferroportin, effectively controlling iron metabolism. Contrary to humans, where a single hepcidin exists, many fish have two functionally distinct hepcidin types, despite having a single ferroportin gene. This raises the question of whether ferroportin is similarly regulated by the iron regulator Hamp1 and the antimicrobial Hamp2. In sea bass (Dicentrarchus labrax), iron overload prompted a downregulation of ferroportin, associated with an upregulation of hamp1, whereas an opposite response was observed during anemia, with no changes in hamp2 in either situation. During infection, ferroportin expression decreased, indicating iron withholding to avoid microbial proliferation. In vivo administration of Hamp1 but not Hamp2 synthetic peptides caused significant reduction in ferroportin expression, indicating that in teleost fish with two hepcidin types, ferroportin activity is mediated through the iron-regulator Hamp1, and not through the dedicated antimicrobial Hamp2. Additionally, in vitro treatment of mouse macrophages with fish Hamp1 but not Hamp2 caused a decrease in ferroportin levels. These results raise questions on the evolution of hepcidin and ferroportin functional partnership and open new possibilities for the pharmaceutical use of selected fish Hamp2 hepcidins during infections, with no impact on iron homeostasis.
Hypoxia-inducible factors (HIFs) are central mediators of cellular adaption to hypoxia. The heterodimeric HIF transcription factors consist of HIF-α and HIF-β, that form functional HIFs. Mammals contain HIF-1α, HIF-2α, and HIF-3α. HIFs play a key role in iron metabolism by regulating the expression of iron-related proteins, such as divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), duodenal cytochrome b (Dcytb), and transferrin receptor (TfR). Hepcidin and iron regulatory proteins (IRPs), the central mediators for systematic and intracellular iron homeostasis, are also regulated by HIFs. In this review, we summarized the regulatory effects of HIFs on iron-related proteins, thus providing insights into the control of HIFs as therapeutic strategies for some iron related disorders.
Cystathionine β-synthase (CBS) catalyzes the transsulfuration pathway and contributes, among other functions, to the generation of hydrogen sulfide (H2 S). In view of the exceptionally high expression of CBS in the liver and the common interleukin-6 (IL-6) pathway utilized in the regulatory systems of H2 S and hepcidin, we speculate that CBS is involved in body iron homeostasis. We found that CBS knock-out (CBS(-/-) ) mice exhibited anemia and a significant increase in iron content in the serum, liver, spleen and heart, along with severe damage to the liver, displaying a hemochromatosis-like phenotype. A high level of hepatic and serum hepcidin was also found. A major cause of the systemic iron-overload is the reduced iron utilization due to suppressed erythropoiesis, which is consistent with an increase in IL-6 and the reduced expression of erythropoietin. Importantly, in the liver, an absence of CBS caused both a reduction in the transcriptional factor NRF2 and an upregulation of hepcidin that lead to a decrease in the iron export protein ferroportin 1. The resulting suppression of iron export exacerbates iron retention, causing damage to hepatocytes. Finally, administration of CBS-overexpressing adenovirus into CBS mutant mice could partially reverse the iron-related phenotype. Our findings thus point to a previously unknown, yet critical role of CBS in iron homeostasis of the body, and the liver in particular. It is likely that a hemochromatosis-like phenotype in patients can be induced by aberration not only in the expression of key molecules in the hepcidin pathway, but also in those related to CBS. This article is protected by copyright. All rights reserved.
Genetic medicine applied to the study of hemochromatosis has identified the systemic loop controlling iron homeostasis, centered on hepcidin-ferroportin interaction. Current challenges are to dissect the molecular pathways underlying liver hepcidin synthesis in response to circulatory iron, HFE, TFR2, HJV, TMPRSS6 and BMP6 functions, and to define the major structural elements of hepcidin-ferroportin interaction. We built a first 3D model of human ferroportin structure, using the crystal structure of EmrD, a bacterial drug efflux transporter of the Major Facilitator Superfamily, as template. The model enabled study of disease-associated mutations, and guided mutagenesis experiments to determine the role of conserved residues in protein stability and iron transport. Results revealed novel amino acids that are critical for the iron export function and the hepcidin-mediated inhibition mechanism: for example, tryptophan 42, localized in the extracellular end of the ferroportin pore and involved in both biological functions. Here, we propose a strategy that is not limited to structure analysis, but integrates information from different sources, including human disease-associated mutations and functional in vitro assays. The first major hypothesis of this PhD thesis is that ferroportin resistance to hepcidin relies on different molecular mechanisms that are critical for ferroportin endocytosis, and include at least three fundamental steps: (i) hepcidin binding to ferroportin, (ii) structural reorganization of the N- and C-ter ferroportin lobes, and (iii) ferroportin ubiquitination.