This project aims to understand the molecular basis for regulation of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been 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. We are culturing neurons from Irp null mice to better characterize their metabolic abnormalities. 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 are also working to characterize Chuvash polycythemia in animal models and human subjects with particular emphasis on the underlying molecular pathophysiology. 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.

Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2013
Total Cost
$958,368
Indirect Cost
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State
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Kovtunovych, Gennadiy; Ghosh, Manik C; Ollivierre, Wade et al. (2014) Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice. Blood 124:1522-30
Robinson, Jeffrey C; Graham, Brian B; Rouault, Tracey C et al. (2014) The crossroads of iron with hypoxia and cellular metabolism. Implications in the pathobiology of pulmonary hypertension. Am J Respir Cell Mol Biol 51:721-9
Frey, Avery G; Nandal, Anjali; Park, Jong Hwan et al. (2014) Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proc Natl Acad Sci U S A 111:8031-6
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Zhang, De-Liang; Ghosh, Manik C; Rouault, Tracey A (2014) The physiological functions of iron regulatory proteins in iron homeostasis - an update. Front Pharmacol 5:124
Rouault, Tracey A (2013) Iron metabolism in the CNS: implications for neurodegenerative diseases. Nat Rev Neurosci 14:551-64
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Linehan, W Marston; Rouault, Tracey A (2013) Molecular pathways: Fumarate hydratase-deficient kidney cancer--targeting the Warburg effect in cancer. Clin Cancer Res 19:3345-52
Rouault, Tracey A; Tong, Wing-Hang (2009) Tangled up in red: intertwining of the heme and iron-sulfur cluster biogenesis pathways. Cell Metab 10:80-1
Zhang, De-Liang; Hughes, Robert M; Ollivierre-Wilson, Hayden et al. (2009) A ferroportin transcript that lacks an iron-responsive element enables duodenal and erythroid precursor cells to evade translational repression. Cell Metab 9:461-73

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