Abnormal Brain Iron Metabolism in Irp2 Deficient Mice Is Associated with Mild Neurological and Behavioral Impairments

Iron Regulatory Protein 2 (Irp2, Ireb2) is a central regulator of cellular iron homeostasis in vertebrates. Two global knockout mouse models have been generated to explore the role of Irp2 in regulating iron metabolism. While both mouse models show that loss of Irp2 results in microcytic anemia and altered body iron distribution, discrepant results have drawn into question the role of Irp2 in regulating brain iron metabolism. One model shows that aged Irp2 deficient mice develop adult-onset progressive neurodegeneration that is associated with axonal degeneration and loss of Purkinje cells in the central nervous system. These mice show iron deposition in white matter tracts and oligodendrocyte soma throughout the brain. A contrasting model of global Irp2 deficiency shows no overt or pathological signs of neurodegeneration or brain iron accumulation, and display only mild motor coordination and balance deficits when challenged by specific tests. Explanations for conflicting findings in the severity of the clinical phenotype, brain iron accumulation and neuronal degeneration remain unclear. Here, we describe an additional mouse model of global Irp2 deficiency. Our aged Irp2−/− mice show marked iron deposition in white matter and in oligodendrocytes while iron content is significantly reduced in neurons. Ferritin and transferrin receptor 1 (TfR1, Tfrc), expression are increased and decreased, respectively, in the brain from Irp2−/− mice. These mice show impairments in locomotion, exploration, motor coordination/balance and nociception when assessed by neurological and behavioral tests, but lack overt signs of neurodegenerative disease. Ultrastructural studies of specific brain regions show no evidence of neurodegeneration. Our data suggest that Irp2 deficiency dysregulates brain iron metabolism causing cellular dysfunction that ultimately leads to mild neurological, behavioral and nociceptive impairments

Smad1/5 is required for erythropoietin-mediated suppression of hepcidin in mice

Anemia suppresses liver hepcidin expression to supply adequate iron for erythropoiesis. Erythroferrone mediates hepcidin suppression by anemia, but its mechanism of action remains uncertain. The bone morphogenetic protein (BMP)-SMAD signaling pathway has a central role in hepcidin transcriptional regulation. Here, we explored the contribution of individual receptor-activated SMADs in hepcidin regulation and their involvement in erythroferrone suppression of hepcidin. In Hep3B cells, SMAD5 or SMAD1 but not SMAD8, knockdown inhibited hepcidin (HAMP) messenger RNA (mRNA) expression. Hepatocyte-specific double-knockout Smad1 fl/fl ;Smad5 fl/fl ;Cre + mice exhibited ∼90% transferrin saturation and massive liver iron overload, whereas Smad1 fl/fl ;Smad5 fl/wt ;Cre + mice or Smad1 fl/wt ;Smad5 fl/fl ;Cre + female mice with 1 functional Smad5 or Smad1 allele had modestly increased serum and liver iron, and single-knockout Smad5 fl/fl ;Cre + or Smad1 fl/fl ;Cre + mice had minimal to no iron loading, suggesting a gene dosage effect. Hamp mRNA was reduced in all Cre+ mouse livers at 12 days and in all Cre+ primary hepatocytes. However, only double-knockout mice continued to exhibit low liver Hamp at 8 weeks and failed to induce Hamp in response to Bmp6 in primary hepatocyte cultures. Epoetin alfa (EPO) robustly induced bone marrow erythroferrone (Fam132b) mRNA in control and Smad1 fl/fl ;Smad5 fl/fl ;Cre + mice but suppressed hepcidin only in control mice. Likewise, erythroferrone failed to decrease Hamp mRNA in Smad1 fl/fl ;Smad5 fl/fl ;Cre + primary hepatocytes and SMAD1/SMAD5 knockdown Hep3B cells. EPO and erythroferrone reduced liver Smad1/5 phosphorylation in parallel with Hamp mRNA in control mice and Hep3B cells. Thus, Smad1 and Smad5 have overlapping functions to govern hepcidin transcription. Moreover, erythropoietin and erythroferrone target Smad1/5 signaling and require Smad1/5 to suppress hepcidin expression.status: publishe

Activin B induces noncanonical SMAD1/5/8 signaling via BMP type I receptors in hepatocytes: Evidence for a role in hepcidin induction by inflammation in male mice

Induction of the iron regulatory hormone hepcidin contributes to the anemia of inflammation. Bone morphogenetic protein 6 (BMP6) signaling is a central regulator of hepcidin expression in the liver. Recently, the TGF-β/BMPsuperfamily member activin B was implicated in hepcidin induction by inflammation via noncanonical SMAD1/5/8 signaling, but its mechanism of action and functional significance in vivo remain uncertain. Here, we show that low concentrations of activin B, but not activin A, stimulate prolonged SMAD1/5/8 signaling and hepcidin expression in liver cells to a similar degree as canonical SMAD2β signaling, and with similar or modestly reduced potency compared with BMP6. Activin B stimulates hepcidin via classical activin type II receptors ACVR2A and ACVR2B, noncanonical BMP type I receptors activin receptor-like kinase 2 and activin receptorlike kinase 3, and SMAD5. The coreceptor hemojuvelin binds to activin B and facilitates activin B-SMAD1/5/8 signaling. Activin B-SMAD1/5/8 signaling has some selectivity for hepatocyte-derived cells and is not enabled by hemojuvelin in other cell types. Liver activin B mRNA expression is up-regulated in multiple mouse models of inflammation associated with increased hepcidin and hypoferremia, including lipopolysaccharide, turpentine, and heat-killed Brucella abortus models. Finally, the activin inhibitor follistatin-315 blunts hepcidin induction by lipopolysaccharide or B. abortus in mice. Our data elucidate a novel mechanism for noncanonical SMAD activation and support a likely functional role for activin B in hepcidin stimulation during inflammation in vivo

Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1β (IL-1β) decreased serum iron within 6 hours, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1β injections decreased serum iron, increased osseous Fgf23 mRNA and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1β injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wild-type and Col4a3KO mice with CKD, but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD

Irp2 regulates insulin production through iron-mediated Cdkal1-catalyzed tRNA modification

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in β cells. This impairs Fe–S cluster biosynthesis, reducing the function of Cdkal1, an Fe–S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2−/− β cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans

Genetic Evidence that the α5 Helix of the Receiver Domain of PhoB Is Involved in Interdomain Interactions

Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein. Generally, response regulators are made up of a receiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain. The mechanisms by which receiver domains control the activities of their respective output domains are unknown. To address this question for the PhoB protein from Escherichia coli, we have employed two separate genetic approaches, deletion analysis and domain swapping. In-frame deletions were generated within the phoB gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the phoA gene. However, another deletion mutant that contained the C-terminal α-helix of the receiver domain (α5) in addition to the entire output domain was unable to activate transcription of phoA. This result suggests that the α5 helix of the receiver domain interacts with and inhibits the output domain. We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N-terminal ∼85% of CheY's receiver domain to the β5-α5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the α5 helix of its unphosphorylated receiver domain and its output domain

Tissue iron content of aged male <i>Irp2^−/−</i> and WT mice.

<p>Total iron content was determined by inductively-coupled plasma optical emission spectroscopy (ICP-OES). Statistical analysis performed by Student's paired t-test (*<i>p</i><0.05; ***<i>p</i><0.001, mean (µg Fe/mg wet tissue weight) ×100± SEM). Ages of mice: WT, 64–75 weeks; <i>Irp2^−/−</i>, 49–71 weeks.</p