Generation of conditional alleles of the murine Iron Regulatory Protein (IRP)-1 and -2 genes

Central aspects of cellular iron metabolism are controlled by IRP1 and IRP2, which are ubiquitously expressed in mouse organs and cells. Total and constitutive deficiency of both IRPs causes embryonic lethality in the mouse. To bypass the early lethality and to study organ-specific and/or temporal functions of IRP1 and/or IRP2 we generated Irp1 and Irp2 conditional alleles. We used mouse lines where a betaGeo gene trap construct was inserted into the second intron of the Irp1 and the Irp2 gene, generating hypomorphic alleles by interrupting the corresponding open reading frame near the amino-termini. The gene trap cassettes are flanked by Frt sites and were co-inserted with LoxP sites flanking exon 3. Flp-mediated removal of the gene trap construct generates floxed alleles with wildtype functions. For both Irp genes, Cre-assisted deletion of exon 3 generates complete null alleles that, in the case of IRP2, are associated with altered body iron distribution and compromised hematopoiesis. If not removed, the gene trap construct causes partially penetrant embryonic lethality unrelated to IRP deficiency when inserted within the Irp1 but not the Irp2 locus. We discuss the implications for functional genomics in the mouse.     

Recombinations between IRP and cystic fibrosis.

A candidate gene for cystic fibrosis was recently isolated by selective cloning of HpaII-tiny-fragment islands; it maps considerably closer to CF than does MET or D7S8 (pJ3.11), and DNA polymorphisms from this region are in marked disequilibrium with CF. cDNA cloning has shown that this protein has a growth factor-like structure and shows homology to the murine and human proto-oncogene int-1; it is designated IRP (int-1-related protein). DNA sequences from the IRP locus that recognize RFLPs are proving to be highly informative for prenatal diagnosis. We report five crossovers that have been identified which occur either within the IRP locus or between IRP and CF; these recombinants demonstrate that CF maps between the DNA markers D7S8 and KM.19.     

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 IRP2_Δ73 (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, IRP2_Δ73 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 IRP2_Δ73-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.     

Blotting analysis of native IRP1: a novel approach to distinguish the different forms of IRP1 in cells and tissues

Iron regulatory protein 1 (IRP1) is a bifunctional protein, which either has aconitase activity or binds to specific mRNA structures to regulate the expression of iron proteins. Using recombinant human IRP1, we found that the two functional forms are resolved by nondenaturing polyacrylamide gel electrophoresis and that they are distinguished from IRP1/RNA complexes. This allowed us to use specific antibodies to develop a blotting system that recognized the iron-free and iron-containing IRP1 forms in the soluble fraction and the RNA-bound IRP1 in the high-speed precipitate fraction of cell extracts. The system was used to study IRP1 in HeLa, K562 cells, and monocytes/macrophages before and after treatment with iron salts, iron chelators, or hydrogen peroxide, as well as in stomach and duodenum biopsies. The results showed that iron-bound aconitase IRP1 is by far the prevalent form in most cells and that the major effect of cellular iron modifications is a shift between free and RNA-bound IRP1. The fraction of RNA-bound IRP1 was highly variable among different cells and was often a minor one. Furthermore, blotting showed that electrophoretic mobility shift assay, as commonly used, tends to under-evaluate the amount of total IRP1 and to over-evaluate the actual RNA-binding activity of IRP1. In conclusion, blotting analysis of IRP1 is a new, useful, and convenient method to analyze the amount and conformations of the protein that reveals previously undetected differences in IRP1 compartmentalization among various cell types.     

HOIL-1 is not required for iron-mediated IRP2 degradation in HEK293 cells

Iron regulatory protein 2 (IRP2) binds to iron-responsive elements (IREs) to regulate the translation and stability of mRNAs encoding several proteins involved in mammalian iron homeostasis. Increases in cellular iron stimulate the polyubiquitylation and proteasomal degradation of IRP2. One study has suggested that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) binds to a unique 73-amino acid (aa) domain in IRP2 in an iron-dependent manner to regulate IRP2 polyubiquitylation and degradation. Other studies have questioned the role of the 73-aa domain in iron-dependent IRP2 degradation. We investigated the potential role of HOIL-1 in the iron-mediated degradation of IRP2 in human embryonic kidney 293 (HEK293) cells. We found that transiently expressed HOIL-1 and IRP2 interact via the 73-aa domain, but this interaction is not iron-dependent, nor does it enhance the rate of IRP2 degradation by iron. In addition, stable expression of HOIL-1 does not alter the iron-dependent degradation or RNA-binding activity of endogenous IRP2. Reduction of endogenous HOIL-1 by siRNA has no affect on the iron-mediated degradation of endogenous IRP2. These data demonstrate that HOIL-1 is not required for iron-dependent degradation of IRP2 in HEK293 cells, and suggest that a HOIL-1 independent mechanism is used for IRP2 degradation in most cell types.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

S-nitrosylation of IRP2 regulates its stability via the ubiquitin-proteasome pathway

Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.     

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.     

Differential translational regulation of IRE-containing mRNAs in Drosophila melanogaster by endogenous IRP and a constitutive human IRP1 mutant

Insects, like vertebrates, express iron regulatory proteins (IRPs) that may regulate proteins in cellular iron storage and energy metabolism. Two mRNAs, an unspliced form of ferritin H mRNA and succinate dehydrogenase subunit b (SDHb) mRNA, are known to comprise an iron responsive element (IRE) in their 5'-untranslated region making them susceptible to translational repression by IRPs at low iron levels. We have investigated the effect of wild-type human IRP1 (hIRP1) and the constitutively active mutant hIRP1-S437 in transgenic Drosophila melanogaster. Endogenous Drosophila IRE-binding activity was readily detected in gel retardation assays. However, translational repression assessed by polysome gradients was only visible for unspliced IRE-containing ferritin H mRNA, but not for SDHb mRNA. Upon expression of exogenous hIRP1-S437 both mRNAs were strongly repressed. This correlated with a diminished survival rate of adult flies with hIRP1 and complete lethality with hIRP1-S437. We conclude that constitutive IRP1 expression is deleterious to fly survival, probably due to the essential function of SDHb or proteins encoded by yet unidentified target mRNAs.