Inherited Disorders of Iron Overload

Dietary iron absorption and systemic iron traffic are tightly controlled by hepcidin, a liver-derived peptide hormone. Hepcidin inhibits iron entry into plasma by binding to and inactivating the iron exporter ferroportin in target cells, such as duodenal enterocytes and tissue macrophages. Hepcidin is induced in response to increased body iron stores to inhibit further iron absorption and prevent iron overload. The mechanism involves the BMP/SMAD signaling pathway, which triggers transcriptional hepcidin induction. Inactivating mutations in components of this pathway cause hepcidin deficiency, which allows inappropriately increased iron absorption and efflux into the bloodstream. This leads to hereditary hemochromatosis (HH), a genetically heterogenous autosomal recessive disorder of iron metabolism characterized by gradual buildup of unshielded non-transferrin bound iron (NTBI) in plasma and excessive iron deposition in tissue parenchymal cells. The predominant HH form is linked to mutations in the HFE gene and constitutes the most frequent genetic disorder in Caucasians. Other, more severe and rare variants are caused by inactivating mutations in HJV (hemojuvelin), HAMP (hepcidin) or TFR2 (transferrin receptor 2). Mutations in SLC40A1 (ferroportin) that cause hepcidin resistance recapitulate the biochemical phenotype of HH. However, ferroportin-related hemochromatosis is transmitted in an autosomal dominant manner. Loss-of-function ferroportin mutations lead to ferroportin disease, characterized by iron overload in macrophages and low transferrin saturation. Aceruloplasminemia and atransferrinemia are further inherited disorders of iron overload caused by deficiency in ceruloplasmin or transferrin, the plasma ferroxidase and iron carrier, respectively

Iron induces insulin resistance in cardiomyocytes via regulation of oxidative stress

York University Librarie

Accelerated CCl4-Induced Liver Fibrosis in Hjv-/- Mice, Associated with an Oxidative Burst and Precocious Profibrogenic Gene Expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl4. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis

Iron-dependent degradation of IRP2 requires its C-terminal region and IRP structural integrity

<p>Abstract</p> <p>Background</p> <p>Iron regulatory protein 2 (IRP2), a post-transcriptional regulator of cellular iron metabolism, undergoes iron-dependent degradation via the ubiquitin-proteasome pathway. A stretch of 73 amino acids within the N-terminal domain 1 of the protein was reported to function as an iron sensor. However, mutants lacking this fragment remain sensitive to degradation in iron-replete cells.</p> <p>Results</p> <p>To identify elements within IRP2 involved in the control of its stability, we undertook a systematic mutagenesis approach. Truncated versions of IRP2 were expressed in H1299 cells and analyzed for their response to iron. Deletion mutants lacking the entire C-terminal domain 4 (amino acids 719–963) of IRP2 remained stable following iron treatments. Moreover, the replacement of domain 4 of IRP1 with the corresponding region of IRP2 sensitized the chimerical IRP1_1–3/IRP2₄protein to iron-dependent degradation, while the reverse manipulation gave rise to a stable chimerical IRP2_1–3/IRP1₄protein. The deletion of just 26 or 34 C-terminal amino acids stabilized IRP2 against iron. However, the fusion of C-terminal IRP2 fragments to luciferase failed to sensitize the indicator protein for degradation in iron-loaded cells.</p> <p>Conclusion</p> <p>Our data suggest that the C-terminus of IRP2 contains elements that are necessary but not sufficient for iron-dependent degradation. The functionality of these elements depends upon the overall IRP structure.</p

Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2Δ73 (lacking a specific insert of 73 amino acids), were injected subcutaneously into nude mice. The induction of IRP2 profoundly stimulated the growth of tumor xenografts, and this response was blunted by addition of tetracycline in the drinking water of the animals, to turnoff the IRP2 transgene. Interestingly, IRP2Δ73 failed to promote tumor growth above control levels. As expected, xenografts expressing the IRP2 transgene exhibited high levels of transferrin receptor 1 (TfR1); however, the expression of other known IRP targets was not affected. Moreover, these xenografts manifested increased c-MYC levels and ERK1/2 phosphorylation. A microarray analysis identified distinct gene expression patterns between control and tumors containing IRP2 or IRP1 transgenes. By contrast, gene expression profiles of control and IRP2Δ73-related tumors were more similar, consistently with their growth phenotype. Collectively, these data demonstrate an apparent pro-oncogenic activity of IRP2 that depends on its specific 73 amino acids insert, and provide further evidence for a link between IRPs and cancer biology

Function of the hemochromatosis protein HFE: Lessons from animal models

Hereditary hemochromatosis (HH) is caused by chronic hyperabsorption of dietary iron. Progressive accumulation of excess iron within tissue parenchymal cells may lead to severe organ damage. The most prevalent type of HH is linked to mutations in the HFE gene, encoding an atypical major histocompatibility complex classImolecule. Shortly after its discovery in 1996, the hemochromatosis protein HFE was shown to physically interact with transferrin receptor 1 (TfR1) and impair the uptake of transferrin-bound iron in cells. However, these findings provided no clue why HFE mutations associate with systemic iron overload. It was later established that all forms of HH result from misregulation of hepcidin expression. This liver-derived circulating peptide hormone controls iron efflux from duodenal enterocytes and reticuloendothelial macrophages by promoting the degradation of the iron exporter ferroportin. Recent studies with animal models of HH uncover a crucial role of HFE as a hepatocyte iron sensor and upstream regulator of hepcidin. Thus, hepatocyte HFE is indispensable for signaling to hepcidin, presumably as a constituent of a larger iron-sensing complex. A working model postulates that the signaling activity of HFE is silenced when the protein is bound to TfR1. An increase in the iron saturation of plasma transferrin leads to displacement of TfR1 from HFE and assembly of the putative iron-sensing complex. In this way, iron uptake by the hepatocyte is translated into upregulation of hepcidin, reinforcing the concept that the liver is the major regulatory site for systemic iron homeostasis, and not merely an iron storage depot

Conditional Derepression of Ferritin Synthesis in Cells Expressing a Constitutive IRP1 Mutant

Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1(C437S) in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1(C437S) in a tetracycline-inducible manner. As expected, IRP1(C437S) stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1(C437S)-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis

Hepcidin Therapeutics

Hepcidin is a key hormonal regulator of systemic iron homeostasis and its expression is induced by iron or inflammatory stimuli. Genetic defects in iron signaling to hepcidin lead to &#8220;hepcidinopathies&#8222; ranging from hereditary hemochromatosis to iron-refractory iron deficiency anemia, which are disorders caused by hepcidin deficiency or excess, respectively. Moreover, dysregulation of hepcidin is a pathogenic cofactor in iron-loading anemias with ineffective erythropoiesis and in anemia of inflammation. Experiments with preclinical animal models provided evidence that restoration of appropriate hepcidin levels can be used for the treatment of these conditions. This fueled the rapidly growing field of hepcidin therapeutics. Several hepcidin agonists and antagonists, as well as inducers and inhibitors of hepcidin expression have been identified to date. Some of them were further developed and are currently being evaluated in clinical trials. This review summarizes the state of the art