This project aims to understand the molecular basis for regulation of intracellular iron metabolism. The RNA features recognized by proteins that mediate the iron-dependent alterations in abundance of ferritin and the transferrin receptor were identified and characterized in previous years in this laboratory. Iron- responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have previously cloned, expressed, and characterized two essential iron- sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2). IRPs bind IREs when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and other transcripts that contain an IRE in the 5' untranslated regions, or in stabilization of the transferrin receptor mRNA and possibly other transcripts that contain IREs in the 3'UTR. The IRE-binding activity of IRP1 depends on whether the protein contains an iron-sulfur cluster (see project HD008814-01). IRP2 also binds IREs in iron-depleted cells, but unlike IRP1, IRP2 is degraded in cells that are iron- replete. There are nine major IRE-containing mRNAs, and many have very important functions, such as the iron exporter, ferroportin, and the oxygen sensor, HIF2 alpha. We discovered that one alternatively spliced transcript of the iron exporter, ferroportin, lacks an IRE, and expression of the non-IRE form in duodenal mucosa and erythroblasts explains several important aspects of physiology. In iron-replete cells, IRP2 is selectively ubiquitinated by FBXL5 and degraded by the proteasome. To approach questions about the physiology of iron metabolism, loss of function mutations of IRP1 and IRP2 have been generated in mice through homologous recombination in embryonic cell lines. In the absence of provocative stimuli, there are subtle abnormalities in iron metabolism associated with loss of IRP1 function. IRP2-/- mice develop a progressive neurologic syndrome characterized by gait abnormalities and axonal degeneration. Ferritin over-expression occurs in affected neurons, and in protrusions of oligodendrocytes into the space created by axonal degeneration. In animals that lack IRP1, IRP 2 compensates for loss of IRP1 regulatory activity, whereas animals that null for both IRP1 and IRP2 die as early embryos. The adult-onset neurodegeneration of adult IRP2-/- mice is exacerbated when one copy of IRP1 is also deleted. IRP2-/- mice offer a unique example of spontaneous adult-onset slowly progressive neurodegeneration, and analyses of gene expression and iron status at various stages of disease are ongoing. We have found that lower motor neurons are very adversely affected, developing axonopathy and death. In addition, small molecule treatment with the stable nitroxide, Tempol, prevents neurodegeneration in IRP2-/- animals. We characterized metabolism in an HLRCC cell line and discovered that AMPK is down, which leads to reduced p53 and DMT1, an iron importer. The iron deficiency that arises as a consequence promotes the switch to aerobic glycolysis. Only HIF1 alpha is significantly elevated, whereas HIF2 alpha expression is repressed by IRP activation. These metabolic changes lead to high storage of glycogen and fatty acids, which enables these cancer cells to store large amounts of energy that may fuel them during when they metastasize and temporarily lose access to nutrients. We discovered that treatment of cells with metformin in combination with an experimental drug that interferes with vascular growth eliminates growth of mouse xenograft tumors formed from the HLRCC cell line. We discovered that Irp1-/- mice develop erythropoietin-driven polycythemia and pulmonary hypertension, and Irp1 is important for modulating expression of HIF2 alpha in pulmonary endothelia and renal interstitial fibroblasts by suppressing translation of the transcript by binding to the 5' iron-responsive element in the transcript. These insights led us to study Chuvash polycythemia, as Chuvash polycythemia is caused by a mutation in the vHL gene that decreases recognition and degradation of hypoxia inducible factors by the vHL complex. Having discovered that Irp1 is important for translation repression of HIF2 alpha, we treated a Chuvash knockin mouse model with TEMPOL, and we reversed polycythemia. Crossing with Irp1-/- mice proved that activation of IRE binding of Irp1 by TEMPOL was crucial to reducing erythropoietin production in these mice, and demonstrated that dietary TEMPOL is a good candidate as a treatment for Chuvash polycythemia. We discovered that the iron exporter, ferroportin, is unexpectedly highly expressed in mature red cells, where it exports free iron released by oxidation of heme. We generated mice with a tissue specific loss of ferroportin in erythroid cells. In cells that lacked ferroportin, oxidative damage shortened the life-span of mature red cells and caused a mild microcytic anemia. Conversely, when the exporter has a gain of function mutation, Q248H, clinical studies with our collaborators showed that the Q248 mutation protects from malarial infection by reducing iron available for nutrition of malarial parasites that infect red cells. The mutation is common in malarious regions of Africa (up to 20%) and in African Americans, where its prevalence ranges from 8-12% of African americans. The mutation likely predisposes to iron overload in tissues such as the liver, kidney and pancreas. We are initiating studies on the iron status and potential deleterious effects on carriers of the mutation by evaluating patients and by making a knocking mouse model, supported by an NIH Bench to Bedside award. Continuing our attempts to find a therapy for HMOX1-/- patients, we infused macrophages cultured in vitro into congenial Hmox1-/- mice, and we observed that macrophage infusion prevented disease, and macrophages multiplied in the liver, where they were became more numerous than in wild type mice. These studies indicated that HMOX1-/- patients, a rare disease, might one day be treatable by correcting their mutations in macrophages removed from patients using CRISPR techniques, and infusion of macrophages that express normal HMOX1 could prevent patients from succumbing to the fatal disease that lack of HMOX1 causes. We identified genes that are induces to express upon erythrophagocytosis, and we are analyzing the metabolic remodeling that permits erythrophagocytosis to proceed without resulting in cell death.

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34
Fiscal Year
2018
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U.S. National Inst/Child Hlth/Human Dev
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Zhang, De-Liang; Rouault, Tracey A (2018) How does hepcidin hinder ferroportin activity? Blood 131:840-842
Zhang, De-Liang; Wu, Jian; Shah, Binal N et al. (2018) Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk. Science 359:1520-1523
Ghosh, Manik C; Zhang, De-Liang; Ollivierre, Hayden et al. (2018) Translational repression of HIF2? expression in mice with Chuvash polycythemia reverses polycythemia. J Clin Invest 128:1317-1325
Rouault, Tracey A; Maio, Nunziata (2017) Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways. J Biol Chem 292:12744-12753
Maio, Nunziata; Kim, Ki Soon; Singh, Anamika et al. (2017) A Single Adaptable Cochaperone-Scaffold Complex Delivers Nascent Iron-Sulfur Clusters to Mammalian Respiratory Chain Complexes I-III. Cell Metab 25:945-953.e6
Maio, N; Rouault, T A (2016) Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20. Metallomics 8:1032-1046
Holmes-Hampton, Gregory P; Crooks, Daniel R; Haller, Ronald G et al. (2016) Use of antisense oligonucleotides to correct the splicing error in ISCU myopathy patient cell lines. Hum Mol Genet 25:5178-5187
Saxena, Neetu; Maio, Nunziata; Crooks, Daniel R et al. (2016) SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery. J Natl Cancer Inst 108:
Rouault, Tracey A (2016) Mitochondrial iron overload: causes and consequences. Curr Opin Genet Dev 38:31-37
Cloonan, Suzanne M; Glass, Kimberly; Laucho-Contreras, Maria E et al. (2016) Mitochondrial iron chelation ameliorates cigarette smoke-induced bronchitis and emphysema in mice. Nat Med 22:163-74

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