Iron Homeostasis Pathways Research Articles

The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 521.

Comparative transcriptome analysis of two rice genotypes differing in their tolerance to saline-alkaline stress

Saline-alkaline stress is an abiotic stress that suppresses rice plant growth and reduces yield. However, few studies have investigated the mechanism by which rice plants respond to saline-alkaline stress at a global transcriptional level. Dongdao-4 and Jigeng-88, which differ in their tolerance to saline-alkaline stress, were used to explore gene expression differences under saline-alkaline stress by RNA-seq technology. In seedlings of Dongdao-4 and Jigeng-88, 3523 and 4066 genes with differential levels of expression were detected, respectively. A total of 799 genes were upregulated in the shoots of both Dongdao-4 and Jigeng-88, while 411 genes were upregulated in the roots of both genotypes. Among the downregulated genes in Dongdao-4 and Jigeng-88, a total of 453 and 372 genes were found in shoots and roots, respectively. Gene ontology (GO) analysis showed that upregulated genes were enriched in several GO terms such as response to stress, response to jasmonic acid, organic acid metabolic process, nicotianamine biosynthetic process, and iron homeostasis. The downregulated genes were enriched in several GO terms, such as photosynthesis and response to reactive oxygen species. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that Dongdao-4 seedlings were specifically enriched in the biosynthesis of secondary metabolites such as diterpenoids and phenylpropanoids. The upregulated genes that were involved in secondary metabolite biosynthesis, amino acid biosynthesis, betalain biosynthesis, organic acid metabolic process, and iron homeostasis pathways may be central to saline-alkaline tolerance in both rice genotypes. In contrast, the genes involved in the diterpenoid and phenylpropanoid biosynthesis pathways may contribute to the greater tolerance to saline-alkaline stress in Dongdao-4 seedlings than in Jigeng-88. These results suggest that Dongdao-4 was equipped with a more efficient mechanism involved in multiple biological processes to adapt to saline-alkaline stress.

Abstract 6073: The lipogenic regulator SREBP induces Transferrin in circulating melanoma cells, suppressing their susceptibility to ferroptosis

Abstract Circulating tumor cells (CTCs) are shed by cancers into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastatic outgrowth. Clonally derived cultured CTCs from patients with BRAF-mutant melanoma reveal upregulation of lipogenesis and iron homeostasis pathways, correlated with their baseline and acquired drug resistance. In CTCs, the lipogenesis regulator SREBP directly induces transcription of the iron carrier Transferrin (TF), thereby reducing intracellular reactive oxygen species (ROS) and lipid peroxidation, and conferring resistance to BRAF inhibitors and inducers of ferroptosis. Knockdown of endogenous TF impairs tumorigenesis by melanoma CTCs; their associated soft agar clonogenic defect is rescued by the lipophilic anti-oxidants Ferrostatin-1 or Vitamin E, and by cholesterol. Single cell RNA-seq of patient-derived melanoma CTCs identifies a subset with high lipogenic, iron metabolic and proliferative signatures, correlated with adverse clinical outcome. Together, the coordinated regulation of these SREBP-driven pathways contributes to cancer progression, drug resistance and metastasis. Citation Format: Xin Hong, Whijae Roh, Ryan J. Sullivan, Keith H. Wong, Ben S. Wittner, HongShan Guo, Taronish D. Dubash, Moshe Sade-Feldman, Ben K. Wesley, Genevieve M. Boland, Dieuwke L. Marvin, Todd Bonesteel, Chenyue Lu, Elad Horwitz, François Aguet, Samuel S. Freeman, Katherine Calhoun, Michelle K. Jewett, Linda T. Nieman, Nir Hacohen, Anders M. Näär, David T. Ting, Mehmet Toner, Shannon L. Stott, Gad Getz, Shyamala Maheswaran, Daniel A. Haber. The lipogenic regulator SREBP induces Transferrin in circulating melanoma cells, suppressing their susceptibility to ferroptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6073.

An Omics Study of Iron and Zinc Homeostasis in Finger Millet: Biofortified Foods for Micronutrient Deficiency in an Era of Climate Change?

The future of food and sustainability of the staple food crops are of utmost importance in the 21st century. Micronutrient deficiency, for example, in iron and zinc, is a common cause of human diseases. Mineral content of the staple food crops has therefore crosscutting importance for food engineering and planetary health. Finger millet, a staple food of agricultural importance worldwide, is rich in iron and zinc, and an ideal model to study the prospects of biofortified foods in times of climate change. We report here a multiomics study of the iron and zinc homeostasis in the finger millet. We identified and characterized 15 candidate genes potentially involved in iron and zinc homeostasis pathways in the finger millet. Structural and functional annotation of the candidate genes revealed a high similarity index with their respective homologs (Oryza sativa, Triticum aestivum, Zea mays, Hordeum vulgare, and Setaria italica). Transcriptome-wide expression analysis showed that genes involved in uptake and translocation of iron and zinc are highly expressed in the GP-1 genotype, while those involved in bioavailability of iron and zinc are expressed more in the GP-45 genotype of the finger millet. In conclusion, finger millet, being a stress-resilient crop, utilizes a combination of strategies in iron and zinc homeostasis pathway, which appear to play an important role in food crop acquisition of iron and zinc, despite environmentally limiting conditions. These data offer molecular insights on iron and zinc accumulation and paves the way for new strategies toward staple food crop with mineral biofortification.

Genetic Variability in the Iron Homeostasis Pathway and Patient Outcomes After Aneurysmal Subarachnoid Hemorrhage.

Iron can be detrimental to most tissues both in excess and in deficiency. The brain in particular is highly susceptible to the consequences of excessive iron, especially during blood brain barrier disruption after injury. Preliminary evidence suggests that iron homeostasis is important during recovery after neurologic injury; therefore, the exploration of genetic variability in genes involved in iron homeostasis is an important area of patient outcomes research. The purpose of this study was to examine the relationship between tagging single nucleotide polymorphisms (SNPs) in candidate genes related to iron homeostasis and acute and long-term patient outcomes after aneurysmal subarachnoid hemorrhage (aSAH). This study was a longitudinal, observational, candidate gene association study of participants with aSAH that used a two-tier design including tier 1 (discovery, n = 197) and tier 2 (replication, n = 277). Participants were followed during the acute outcome phase for development of cerebral vasospasm and delayed cerebral ischemia (DCI) and during the long-term outcome phase for death and gross functional outcome using the Glasgow Outcome Scale (GOS; poor = 1-3). Genetic association analyses were performed using a logistic regression model adjusted for age, sex, and Fisher grade. Approximate Bayes factors (ABF) and Bayesian false discovery probabilities (BFDP) were used to prioritize and interpret results. In tier 1, 235 tagging SNPs in 28 candidate genes were available for analysis and 26 associations (20 unique SNPs in 12 genes) were nominated for replication in tier 2. In tier 2, we observed an increase in evidence of association for three associations in the ceruloplasmin (CP) and cubilin (CUBN) genes. We observed an association of rs17838831 (CP) with GOS at 3months (tier 2 results, odds ratio [OR] = 2.10, 95% confidence interval [CI] = 1.14-3.86, p = 0.018, ABF = 0.52, and BFDP = 70.8%) and GOS at 12months (tier 2 results, OR = 1.86, 95% CI 0.98-3.52, p = 0.058, ABF = 0.72, and BFDP = 77.3%) as well as rs10904850 (CUBN) with DCI (tier 2 results, OR = 0.70, 95% CI 0.48-1.02, p = 0.064, ABF = 0.59, and BFDP = 71.8%). Among the genes examined, our findings support a role for CP and CUBN in patient outcomes after aSAH. In an effort to translate these findings into clinical utility and improve outcomes after aSAH, additional research is needed to examine the functional roles of these genes after aSAH.

Profile of Mary Lou Guerinot

On the first Earth Day, April 22, 1970, Mary Lou Guerinot decided that she would major in biology in college in the hope of promoting environmental sustainability. As the 50th anniversary of Earth Day approaches, Guerinot, now a professor of biological sciences at Dartmouth College and a member of the National Academy of Sciences, is well on her way toward achieving that goal through her work on the molecular mechanisms of metal ion uptake and its regulation. Guerinot’s work is laying the foundation for environmentally sustainable, nutrient-dense crops, as well as plant-based solutions for removal of toxic metals from soil. Her Inaugural Article (1) reports the identification of a transcription factor essential for plant growth under iron deficiency. The finding moves her team closer to understanding the iron homeostasis pathway in plants, holding promise for improving agricultural productivity and human health. Mary Lou Guerinot. Image courtesy of Olga Zhaxybayeva (Dartmouth College, Hanover, NH). Guerinot was raised in upstate New York, where she attended the Rochester-based Catholic all-girls St. Agnes High School. “Unlike the boys’ Catholic high school nearby, it was basically science-light,” she says. “We didn’t have physics or calculus, and very few students went on to college to study science.” She and a few other students urged their math teacher to let them study calculus on their own. The hard work on this subject and others paid off, and Guerinot was accepted at Cornell University’s College of Agriculture and Life Sciences. She adhered to her decision to major in biology and earned a Bachelor of Science degree with distinction at Cornell in 1975. Four years later, Guerinot earned a doctorate in biology from Dalhousie University under the direction of David Patriquin, studying the sea urchin–lobster–kelp ecosystem. Envisioning life as a marine microbiologist, Guerinot did a postdoctoral fellowship at the …

Iron homeostasis, complement, and coagulation cascade as CSF signature of cortical lesions in early multiple sclerosis.

ObjectiveIntrathecal inflammation, compartmentalized in cerebrospinal fluid (CSF) and in meningeal infiltrates, has fundamental role in inflammation, demyelination, and neuronal injury in cerebral cortex in multiple sclerosis (MS). Since the exact link between intrathecal inflammation and mechanisms of cortical pathology remains unknown, we aimed to investigate a detailed proteomic CSF profiling which is able to reflect cortical damage in early MS.MethodsWe combined new proteomic method, TRIDENT, CSF analysis, and advanced 3T magnetic resonance imaging (MRI), in 64 MS patients at the time of diagnosis and 26 controls with other neurological disorders. MS patients were stratified according to cortical lesion (CL) load.ResultsWe identified 227 proteins differently expressed between the patients with high and low CL load. These were mainly related to complement and coagulation cascade as well as to iron homeostasis pathway (30 and 6% of all identified proteins, respectively). Accordingly, in the CSF of MS patients with high CL load at diagnosis, significantly higher levels of sCD163 (P < 0.0001), free hemoglobin (Hb) (P < 0.05), haptoglobin (P < 0.0001), and fibrinogen (P < 0.01) were detected. By contrast, CSF levels of sCD14 were significantly (P < 0.05) higher in MS patients with low CL load. Furthermore, CSF levels of sCD163 positively correlated (P < 0.01) with CSF levels of neurofilament, fibrinogen, and B cell‐related molecules, such as CXCL13, CXCL12, IL10, and BAFF.InterpretationIntrathecal dysregulation of iron homeostasis and coagulation pathway as well as B‐cell and monocyte activity are strictly correlated with cortical damage at early disease stages.

Saccharomyces cerevisiae as a viable model for the overexpression and purification of proteins involved in iron homeostasis

Iron is indispensable for the eukaryotic cell but excess of iron is toxic. Disruptions in iron metabolism leads to numerous human diseases, hence the study of iron regulation and metabolism is of high importance. Saccharomyces cerevisiae is used as a model system in our lab to study iron trafficking and homeostasis pathways to better understand iron regulation at the cellular and molecular level. In yeast, two paralogous transcriptional activators, Aft1 and Aft2, play a central role in iron regulation by activating the transcription of target genes in response to iron deprivation. Monothiol glutaredoxins Grx3 and Grx4 that utilize glutathione to bind [2Fe‐2S] clusters interact specifically with Aft1 and Aft2, transferring an Fe‐S cluster to Aft1/2 that inhibits its DNA binding activity. Aft1 is considered the primary regulator of iron homeostasis because aft1Δ mutants exhibit a stronger iron deficiency phenotype than aft2Δ mutants. Aft1 and Aft2 share 39% sequence homology in their N‐terminal DNA binding domains. The only available structure is of a truncated version of Aft2 that contains this homologous portion and includes the domains responsible for DNA binding and iron sensing. Aft1/2 have been shown to be very unstable and difficult to overexpress and purify in Escherichia coli. Hence we optimized the growth conditions to overexpress Aft1 in its native organism, Saccharomyces cerevisiae, and further try to purify Aft1 from the same. Further studies will be carried out in order to investigate the in‐vitro interaction between full length Aft1/2 and Grx3/4‐Fra2 to better understand the mechanism of Aft1/2 inhibition at the molecular level.Support or Funding InformationSupported by Dr. Caryn E. Outten's Research FundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Iron Homeostasis in Rice: Deficit and Excess

Rice is the major food crop grown in all over the world mitigating 60–70% requirement of calories. However, its growth has always been challenged under different abiotic stress. Iron is one such important micronutrient, which pose hazardous effects to its yield and quality under both iron (Fe) deficient and excess condition. Recent research studies have attempted to overcome these iron stress constraints by understanding the function of homeostatic genes involved in it and modulating their expression through developed molecular technologies. These technologies involved overexpression of genes in iron uptake, transport, storage, along with the transcriptomics analysis of whole genome to develop a better quality of rice progenies which can withstand various adverse effects of iron stress. Iron homeostasis pathway involves Strategy I and Strategy II followed by dicotyledonous and monocotyledonous plants. However, in the case of rice, several genes involved in both strategies have found to be responsive under Fe-deficient and excess condition. Interaction of other metals present in the soil along with pH can affect the iron uptake in rice. Transgenic rice can become a strong contender in iron stress condition controlling iron uptake, translocation and storage.

Abstract 2031: Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft

Abstract Introduction: Tumor cells present an iron metabolic disorder with high proliferation rate, increased iron storage (ferritin and Labile Iron Pool - LIP) and high sensibility to iron deprivation, which could be a therapeutic target. Anticancer effect of iron chelators deferoxamine (DFO) and deferasirox (DFX) has been revealed in several types of cancers. In breast cancer (BC), the development of new therapeutic approaches is urgently needed for triple negative (TN) subtype which presents poor prognosis and lacks targeting therapy. We investigated the therapeutic potential of iron chelators combined with chemotherapeutic agents in TNBC cell lines and patient-derived xenografts (PDX). Methods: Anticancer effects of iron chelators combined with chemotherapeutic agents (doxorubicin, cisplatin or carboplatin) were evaluated in vitro in 4 TNBC cell lines by MTT assay, annexin V/PI staining and assessment of caspase 3/7 activity. Assessment of LIP, transferrin receptor 1 (TfR1) expression level, Reactive Oxygen Species (ROS) production and mitochondrial membrane potential variations were performed by flow cytometry, and ELISA assay for ferritin level. The activity of DFX alone or combined with doxorubicin/cyclophosphamide (AC) was tested in the HBCx-10 TNBC PDX selected because of its relapse to AC. Iron homeostasis, hypoxia and PI3K pathway were analyzed by immunohistochemistry (IHC) and Western-blot in both cell lines and PDX tumors. In DFX+/-AC treated and untreated tumors, induction of apoptosis was performed by TUNEL assay and a transcriptome analysis is ongoing. Results: Iron chelators acted in synergy effect with three chemotherapies in all cell lines which were tested to inhibit cell proliferation and induce apoptosis. Chelators increased cytotoxicity until 60% compared to chemotherapies alone. In all cell lines, chelators treatment increased TfR1 expression level and decreased LIP and ferritin. Furthermore, down-regulation of PI3K pathway, hypoxia, mitochondrial membrane potential, and the production increasing of ROS were observed. In HBCx-10 PDX, a trend for antitumoral activity of DFX alone was observed (p=0.09) at the end of the experiment (day 81). A significant difference of Relative Tumor Volume (RTV) was observed since day 18 between the AC group and the DFX+AC group (tumor growth inhibition: 37 to 61%, tumor growth delay: 10 to 14 days, 0.005&amp;lt;p&amp;lt;0.04). Same as the observing in vitro, modulations of PI3K pathway and hypoxia are involved in this antitumoral synergy. Except neutropenia (due to chemotherapy), no other hematologic toxicity was observed in both AC and DFX+AC groups. Conclusions: Iron chelators may increase the effectiveness of conventional chemotherapies for TNBC treatments. This antitumoral synergy involves PI3K pathway downregulation, ROS production and decrease mitochondrial membrane potential. Citation Format: Sandrine Tury, Sophie Vacher, Véronique Becette, Franck Assayag, Sophie Chateau-Joubert, Jean-Luc Servely, Elisabetta Marangoni, Ivan Bièche, Céline Callens. Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2031. doi:10.1158/1538-7445.AM2017-2031

Activated Oncogenic Pathway Modifies Iron Network in Breast Epithelial Cells: A Dynamic Modeling Perspective

Dysregulation of iron metabolism in cancer is well documented and it has been suggested that there is interdependence between excess iron and increased cancer incidence and progression. In an effort to better understand the linkages between iron metabolism and breast cancer, a predictive mathematical model of an expanded iron homeostasis pathway was constructed that includes species involved in iron utilization, oxidative stress response and oncogenic pathways. The model leads to three predictions. The first is that overexpression of iron regulatory protein 2 (IRP2) recapitulates many aspects of the alterations in free iron and iron-related proteins in cancer cells without affecting the oxidative stress response or the oncogenic pathways included in the model. This prediction was validated by experimentation. The second prediction is that iron-related proteins are dramatically affected by mitochondrial ferritin overexpression. This prediction was validated by results in the pertinent literature not used for model construction. The third prediction is that oncogenic Ras pathways contribute to altered iron homeostasis in cancer cells. This prediction was validated by a combination of simulation experiments of Ras overexpression and catalase knockout in conjunction with the literature. The model successfully captures key aspects of iron metabolism in breast cancer cells and provides a framework upon which more detailed models can be built.

Abstract 12691: The Biological Clock Regulates Cellular Iron Homeostasis

Introduction: Iron is an essential metal for various cellular processes including mitochondrial respiration, DNA synthesis and protein synthesis. Low iron stores can disrupt any of these processes, while excess free iron increases oxidative stress. It is thus crucial to regulate iron levels to meet cellular needs while preventing excess iron accumulation. Previously it was reported that human serum iron levels show diurnal variations. However, the underlying mechanism by which iron homeostasis shows rhythmic variations and whether circadian iron regulation also exists at cellular level is unknown. Since the main function of the biological clock is to synchronize cellular demands with environmental signals such as nutrition, we hypothesize that the biological clock regulates cellular iron homeostasis to protect cells from oxidative damage. Results: Assessment of iron levels in various tissues in mice revealed that hepatic iron levels display 24-hour diurnal variations with peaks by the onset of the active period. Furthermore, disruption of the biological clock by the deletion of circadian transcriptional activator Bmal1 caused an increase in hepatic iron levels, which was associated with a decrease in serum iron. To investigate the underlying mechanism of the clock-mediated regulation of cellular iron, we next measured the mRNA levels of iron homeostasis genes in the liver of Bmall -/- and WT mice. Our results indicated that among all proteins involved in iron homeostasis, the levels of the iron exporter, ferroportin-1 (FPN1) was significantly decreased in Bmal -/- mice. Since Bmal1 deletion is associated with an increase in cellular iron, the reduced levels of FPN1 suggests that decreased iron export through FPN1 might be the mechanism for iron accumulation in Bmal -/- livers. Bmal1 deletion was also associated with decreased levels of iron importer protein transferrin-1, consistent with a cellular signature of iron overload. Finally, mRNA expression of FPN1 and TfR1 also displayed diurnal variations. Conclusions: Our data establish a link between the biological clock and cellular iron homeostasis pathways in mammals. Our data also suggests that FPN1 and TfR1 expression might be controlled by the clock machinery.

Metabolomic profiling identifies potential pathways involved in the interaction of iron homeostasis with glucose metabolism

ObjectiveElevated serum ferritin has been linked to type 2 diabetes (T2D) and adverse health outcomes in subjects with the Metabolic Syndrome (MetS). As the mechanisms underlying the negative impact of excess iron have so far remained elusive, we aimed to identify potential links between iron homeostasis and metabolic pathways. MethodsIn a cross-sectional study, data were obtained from 163 patients, allocated to one of three groups: (1) lean, healthy controls (n = 53), (2) MetS without hyperferritinemia (n = 54) and (3) MetS with hyperferritinemia (n = 56). An additional phlebotomy study included 29 patients with biopsy-proven iron overload before and after iron removal. A detailed clinical and biochemical characterization was obtained and metabolomic profiling was performed via a targeted metabolomics approach. ResultsSubjects with MetS and elevated ferritin had higher fasting glucose (p < 0.001), HbA1c (p = 0.035) and 1 h glucose in oral glucose tolerance test (p = 0.002) compared to MetS subjects without iron overload, whereas other clinical and biochemical features of the MetS were not different. The metabolomic study revealed significant differences between MetS with high and low ferritin in the serum concentrations of sarcosine, citrulline and particularly long-chain phosphatidylcholines. Methionine, glutamate, and long-chain phosphatidylcholines were significantly different before and after phlebotomy (p < 0.05 for all metabolites). ConclusionsOur data suggest that high serum ferritin concentrations are linked to impaired glucose homeostasis in subjects with the MetS. Iron excess is associated to distinct changes in the serum concentrations of phosphatidylcholine subsets. A pathway involving sarcosine and citrulline also may be involved in iron-induced impairment of glucose metabolism.

Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.

Kidney-liver dialogue in acute kidney injury

hemopexin is a multifunctional serum glycoprotein that plays important roles in iron homeostasis, antioxidant protection, anti-inflammatory effects, and signal transduction pathways ([6][1], [8][2]). The liver is the main source of the hemopexin synthesis ([7][3]), but expression in other tissues

Genome-wide Association Studies and Human Disease

MANY COMMON COMPLEX DISEASES SUCH AS hypertension, diabetes, coronary heart disease, psychiatric disorders, and some cancers have a genetic etiology. Despite enormous efforts over the last few decades, little real progress was made in finding the genes and causal variants involved. Genome-wide association studies, in which hundreds of thousands of DNA markers are tested (usually in a casecontrol design) for association with disease, provide the first effective approach to search for genetic variants that contribute to the complex etiology of common human diseases. In the last 3 years, almost 1000 variants associated with a range of human traits and common diseases have been identified using genome-wide association methods (FIGURE). To date, most of these studies have been in populations of European descent. Genome-wide search strategies developed from advances in genotyping technology, greater understanding of the structure of common variation in the human genome, and continued advances in computing power and software tools. Commercial genotyping platforms can type as many as 1 million single-nucleotide polymorphisms (SNPs) on a single chip, capturing (tagging) most variation between individuals in a single experiment. Instead of genotyping per sample the 10 million to 15 million common SNPs that segregate in the population for each sample, a much smaller subset of approximately 500 000 SNPs is sufficient to cover common variation in the genome. The flipside of this redundancy is that SNPs that are statistically associated with disease are unlikely to be causal and will be correlated with an ungenotyped causal variant. Genome-wide association studies have provided insights about disease, in particular: (1) for almost any disease that has been investigated, there are SNP variants common in the population (with an allele frequency 5%) that are robustly associated with disease; (2) most of these variants are in genes that contribute to biological pathways that were previously not known to be involved in disease or are nowhere near a known protein-coding gene; (3) the effect sizes of associated SNPs are typically small with odds ratios of risk alleles in the range of approximately 1.1 to 1.5; (4) for any particular disease, accumulating the effects of many different SNPs associated with a disease usually explains only a small fraction of the familial risk (or heritability); and (5) not all diseases and traits are alike in genetic architecture. For example, in age-related macular degeneration, approximately 50% of genetic variation has been accounted for by only 6 loci, whereas for adult height, only 6% of genetic variation has been accounted for by approximately 50 loci. In the iron homeostasis pathway, several common SNPs have been reported that each explain 5% or more of genetic variation. Failure to account for much of the genetic variation or “missing heritability” has divided the community with respect to the success or failure of genome-wide association studies. Since the common goal is to understand pathways to disease and develop improved methods of prevention, diagnosis, and treatment, it is important to understand what the current flood of associated SNPs reveals about disease biology and gene regulation, why so little genetic variation has been accounted for, and what experimental approaches might lead the identification of causal variants and mechanisms. Figure. The Genome-wide Association Revolution: From Trickle to Flood

Effects of the Aspergillus fumigatus siderophore systems on the regulation of macrophage immune effector pathways and iron homeostasis

The saprophytic fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen, which is responsible for invasive aspergillosis in immunocompromised patients. Iron plays an essential role for the growth and proliferation of A. fumigatus. This fungus synthesizes three major siderophores. It excretes triacetylfusarinine C to capture iron, while it accumulates ferricrocin and hydroxyferricrocin for hyphal and conidial iron storage, respectively. Herein, we investigated the role of the siderophore system of A. fumigatus in the modulation of immune effector pathways and iron homeostasis in macrophages. We set up a co-culture system consisting of the murine macrophage cell line RAW264.7 and either A. fumigatus wild type or a siderophore-deficient mutant (Δ sidA). We used real-time quantitative RT-PCR and Western blot analyses to study the expression of macrophage iron metabolism and innate immune response genes in response to pathogen challenge. Infection of macrophages with A. fumigatus wild type, but not with the Δ sidA mutant, induced expression of TNF and phagocyte oxidase subunit 47 at the transcriptional level. Moreover, infection with A. fumigatus wild type, but not with the Δ sidA mutant, compromised macrophage iron homeostasis. Infection with wild-type A. fumigatus decreased expression of the two cellular iron importers, the divalent metal transporter-1 and the transferrin receptor, and the only known iron exporter ferroportin. At the same time, it increased macrophage iron retention and ferritin synthesis. These data indicate that A. fumigatus affects the regulation of macrophage iron homeostasis and innate immune effector pathways via its siderophore system. The changes in immune response may be a consequence of macrophage iron restriction.

Hereditary Hemochromatosis Protein, HFE, Interaction with Transferrin Receptor 2 Suggests a Molecular Mechanism for Mammalian Iron Sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.

Unexpected role of ceruloplasmin in intestinal iron absorption

Ferroxidases are essential for normal iron homeostasis in most organisms. The paralogous vertebrate ferroxidases ceruloplasmin (Cp) and hephaestin (Heph) are considered to have nonidentical functions in iron transport: plasma Cp drives iron transport from tissue stores while intestinal Heph facilitates iron absorption from the intestinal lumen. To clarify the function of Cp, we acutely bled Cp-/- mice to stress iron homeostasis pathways. Red cell hemoglobin recovery was defective in stressed Cp-/- mice, consistent with low iron availability. Contrary to expectations, iron was freely released from spleen and liver stores in Cp-/- mice, but intestinal iron absorption was markedly impaired. Phlebotomy of wild-type mice caused a striking shift of Cp from the duodenal epithelium to the underlying lamina propria, suggesting a critical function of Cp in basolateral iron transport. Regulated relocalization of intestinal Cp may represent a fail-safe mechanism in which Cp shares with Heph responsibility for iron absorption under stress.

The Saccharomyces cerevisiae Sub2 protein suppresses heterochromatic silencing at telomeres and subtelomeric genes

We show that overexpression of Sub2p, a multifunctional Saccharomyces cerevisiae helicase family member that is involved in mRNA elongation and transport, also suppresses heterochromatic silencing at telomeres. Genetic assays show cells that overexpress SUB2 from a high copy plasmid exhibit increased survival rates when selecting for a telomere-silenced URA3 reporter. Two temperature-sensitive sub2 mutations that affect different helicase domains were also examined at the permissive temperature; these mutants also overcome silencing of the URA3 reporter. The degree to which silencing is suppressed correlates with SUB2 RNA and protein levels. Additionally, we find that Sub2p localizes to the telomeres, as determined by chromatin immunoprecipitation assays, suggesting that Sub2p has a direct effect at telomeres. Genome-wide analysis of transcripts was used to assess whether Sub2p overproduction affects only the silenced URA3 reporter gene, or whether other subtelomeric genes are also affected. Of the 70 RNA transcripts elevated in the Sub2p overexpressing cells, 28% are encoded by subtelomeric genes that are located within 5 Kbp of a core X or Y' repeat. The remainder of the transcripts clustered into several functional groups, including the iron homeostasis pathway, purine nucleotide metabolism, and miscellaneous transport genes, among others. These results suggest a targeted effect of Sub2p on transcription. Our results also confirm that Sub2p affects heterochromatic gene expression, similar to that observed with the Drosophila Hel25E homologue. The above observations imply that Sub2p affects chromatin structure in addition to, or in parallel with, its functions in transcription elongation, splicing and mRNA transport.