Expression of iron-related genes in human brain and brain tumors
© Hänninen et al; licensee BioMed Central Ltd. 2009
Received: 05 September 2008
Accepted: 22 April 2009
Published: 22 April 2009
Defective iron homeostasis may be involved in the development of some diseases within the central nervous system. Although the expression of genes involved in normal iron balance has been intensively studied in other tissues, little is known about their expression in the brain. We investigated the mRNA levels of hepcidin (HAMP), HFE, neogenin (NEO1), transferrin receptor 1 (TFRC), transferrin receptor 2 (TFR2), and hemojuvelin (HFE2) in normal human brain, brain tumors, and astrocytoma cell lines. The specimens included 5 normal brain tissue samples, 4 meningiomas, one medulloblastoma, 3 oligodendrocytic gliomas, 2 oligoastrocytic gliomas, 8 astrocytic gliomas, and 3 astrocytoma cell lines.
Except for hemojuvelin, all genes studied had detectable levels of mRNA. In most tumor types, the pattern of gene expression was diverse. Notable findings include high expression of transferrin receptor 1 in the hippocampus and medulla oblongata compared to other brain regions, low expression of HFE in normal brain with elevated HFE expression in meningiomas, and absence of hepcidin mRNA in astrocytoma cell lines despite expression in normal brain and tumor specimens.
These results indicate that several iron-related genes are expressed in normal brain, and that their expression may be dysregulated in brain tumors.
Regulation of iron homeostasis is crucial to maintain normal cell function, and abnormal cellular iron content has been associated with various diseases. Iron is an essential cofactor for many proteins particularly in oxidative reactions and, therefore, neuronal tissues with a high rate of oxidative metabolism have a considerable requirement for iron . The central nervous system (CNS) is not directly in contact with the plasma iron pool, because it resides behind the blood-brain barrier. Like many molecules, the entry of iron is tightly regulated by the blood-brain barrier, and specific transport mechanisms are required to transfer iron into the brain tissue . Several gene products involved in the regulation of iron homeostasis are expressed in the murine CNS, including transferrin receptor 1 (TfR1) , iron regulatory protein , ferritin , neogenin , and hepcidin . Even though iron has an essential role for normal physiology, it poses a threat to cells and tissues when present in excess. This feature is based on iron's ability to readily participate in oxidation-reduction reactions and the formation of reactive oxygen intermediates . Iron accumulation has been reported in the nervous tissue of patients suffering from various neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and age-related macular degeneration [7–10].
Malignant CNS tumors arise worldwide in approximately 189,000 patients per year with an estimated annual mortality of 142,000 . Malignant gliomas, including the most common subtype, glioblastoma, are among the most devastating neoplasms and still pose a great challenge for both diagnosis and treatment. Malignant gliomas derive from glial cells and exhibit aggressive tumor characteristics such as high proliferation rate, diminished apoptosis, and escape from external growth control. A distinctive feature of gliomas is that they very rarely metastasize beyond the CNS, despite their highly invasive and angiogenic capabilities .
In neoplastic tissues, the oxygen requirement is high, and thus angiogenesis is often crucial for cancer survival. For malignant CNS tumors, especially gliomas, neovascularization is directly correlated with their biological aggressiveness, degree of malignancy and clinical recurrence, and is inversely correlated with post-operative survival time . Neovascularization is driven by a complex regulatory pathway involving von Hippel-Lindau protein (pVHL) and hypoxia-inducible factor (HIF). In normoxia, the transcription factor HIF is targeted for proteosomal degradation, whereas it is stabilized in hypoxic conditions. HIF accumulation induces the expression of several proangiogenic proteins, including transferrin and TfR1, that are linked to the regulation of cellular iron homeostasis .
Hepcidin is a key regulator of iron homeostasis that is abundantly expressed in the liver [14, 15]. Iron overload leads to increased hepcidin synthesis, while anaemia, hypoxia, and inflammation downregulate its expression . A recent study showed that hypoxia downregulates hepcidin expression via the VHL/HIF pathway . Hepcidin acts by modulating the activity of ferroportin, the only iron export protein known to exist in mammalian cells. By binding to ferroportin, hepcidin causes its internalization and degradation and, thereby, decreases cellular iron export . Hemojuvelin and its putative co-regulator, neogenin, are other factors that may be involved in the regulation of hepcidin expression [19–21].
Because malignant cells have a high metabolic rate and require significant quantities of iron for various cellular processes, our hypothesis was that iron related genes are differentially expressed in brain tumor cells versus normal brain tissue. The goal of this study was to investigate the expression levels of genes related to iron homeostasis in both brain tumors and normal brain regions. The mRNA expression levels of hepcidin (HAMP), HFE, neogenin (NEO1), transferrin receptor 1 (TFRC), transferrin receptor 2 (TFR2), and hemojuvelin (HFE2) were measured. We found that all the studied genes, except for hemojuvelin, were expressed in normal brain, and altered expression patterns were found in brain tumors and astrocytoma cell lines. The results suggest that these genes might play a role in the maintenance of iron homeostasis in the brain, and their differential expression in brain tumors may contribute to tumor pathogenesis.
Study materials for RNA isolation
Patient information for brain tumor samples
Type of tumor
Age at diagnosis
Spearman's rank correlation coefficient was used to determine the influence of age on expression levels. The results were considered statistically significant when the correlation coefficient was < -0.300 or > 0.300, with p-value < 0.05. Mann-Whitney U test was used to evaluate the effect of sex on the expression levels. The results were considered statistically significant when p-value was < 0.05.
RNA isolation and first-strand cDNA synthesis
All tissue specimens to be used for RNA isolation were placed in RNAlater (Ambion, Austin, TX) and stored at -80°C until use. Total RNA was isolated from tissue samples using QIAzol lysis reagent and RNeasy Lipid Tissue Mini Kit (Qiagen, Germantown, MD) and from cell cultures using RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. Both protocols included treatment with RNase-free DNase I (Novagen, Madison, WI). RNA concentration and purity were determined by optical density measurement at 260 and 280 nm. All the samples had an OD260/OD280 ratio of 2.0 or higher. For each sample, 0.6 μg of total RNA were converted into first-strand cDNA using the First-Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada) and oligo d(T) primers according to the protocol recommended by the manufacturer.
Reverse transcription PCR and quantitative real-time PCR
Primer sequences for quantitative real-time PCR
Forward primer (5'-3')
Reverse promer (5'-3')
PCR reactions were performed using ReddyMix PCR Master Mix (ABgene, UK) according to manufacturer's instructions. B2M (beta-2-microglobulin) was used as positive control for the RT-PCR reactions. The PCR products were visualized by gel electrophoresis in 1.5% agarose gel with 0.1 μL/mL ethidium bromide. Quantitative real-time PCR reactions were carried out in a 20 μL volume containing 0.5 μL of first strand cDNA, 1× of QuantiTect SYBR Green PCR Master Mix (Qiagen, Hilden, Germany), and 0.5 μM of forward and reverse primers. Amplification and detection were carried out as follows. After an initial 15-min activation step at 95°C, 45 cycles consisting of denaturation at 94°C, 15 s; annealing at Tm, 20 s; elongation at 72°C, 20 s, and final cooling step. Melting curve analysis was always performed after the amplification to check PCR specificity. To quantify the concentration of both the internal control transcripts and the transcripts for HAMP, HFE, NEO1, TFRC, and TFR2 in the samples, a standard curve for each transcript was established using 5-fold serial dilutions of known concentrations of purified PCR products generated from the same primer sets. Each cDNA sample was tested in triplicate. Replicate analyses were automatically handled by the software of the real-time PCR machine. The crossing point value obtained was utilized to determine the amount of original transcript using the specific standard curve. The geometric mean of the 4 internal control transcripts was used as a normalization factor for gene expression levels . The final relative mRNA expression was designated as the copy number of a target transcript in each tissue divided by the corresponding normalization factor and subsequently multiplied by 102 or 103.
Results and discussion
In this study, the mRNA expression levels of hepcidin (HAMP), HFE, neogenin (NEO1), TfR1 (TFRC), transferrin receptor 2 (TFR2), and hemojuvelin (HFE2) were examined in normal human brain tissue, brain tumors, and astrocytoma cell lines. Hemojuvelin showed no expression in RT-PCR experiments and, therefore, it was not analyzed further by quantitative real-time PCR.
The statistical analysis revealed no significant influence of either age or sex on the expression levels of genes studied.
Hemojuvelin also plays an important role in hepcidin regulation in the liver, acting as a co-receptor for bone morphogenetic protein . Hemojuvelin exists in both membrane-bound and soluble forms, and Silvestri et al.  have demonstrated regulation of soluble hemojuvelin via HIF-1α and furin. However, our results indicate that hemojuvelin mRNA is not expressed in normal brain tissue or in brain tumors. A previous study demonstrated that murine brain also does not express hemojuvelin . Taken together, the available data suggest that hemojuvelin may not regulate hepcidin expression in the brain, or soluble hemojuvelin produced elsewhere is transferred across the blood-brain barrier to regulate hepcidin expression in the brain.
Although investigation of the molecular basis of iron homeostasis has been intensive after the discovery of the HFE gene in 1996 , little is still known about the role of iron and iron-related genes in the brain. In this study, we show that many of the genes considered essential for the regulation of iron homeostasis outside the CNS are also expressed in the brain. Iron regulation in the brain is complex, and in future studies it will be of great interest to investigate the protein expression of these molecules in different brain regions along with assessment of regional iron concentrations. Due to the presence of the blood-brain barrier, the CNS can adjust its iron status somewhat independently of other organs. Our results also indicate that the expression pattern of iron-related genes can vary markedly in tumors arising within the brain. Further investigation will be required to evaluate whether the dysregulated expression of iron-related genes in brain tumors may contribute to tumor pathogenesis.
The present study describes the expression of several iron-related genes including hepcidin (HAMP), HFE, neogenin(NEO1), transferrin receptor 1 (TFRC), transferrin receptor 2 (TFR2), and hemojuvelin(HFE2) in normal human brain, brain tumors, and astrocytoma cell lines. Our results suggested that all these genes except for HFE2 are expressed in the normal brain, and that their expression may be dysregulated in certain brain tumors.
We thank Mrs. Aulikki Lehmus and Mrs. Reija Randen for skilful technical assistance and Mrs. Anna-Maija Koivisto for assistance with statistical analysis. This work was supported by grants from the Medical Research Fund of Tampere University Hospital and the Sigrid Juselius Foundation.
- Zecca L, Youdim MB, Riederer P, Connor JR, Crichton RR: Iron, brain ageing and neurodegenerative disorders. Nat Rev Neurosci. 2004, 5 (11): 863-873. 10.1038/nrn1537.View ArticlePubMedGoogle Scholar
- Burdo JR, Connor JR: Brain iron uptake and homeostatic mechanisms: an overview. Biometals. 2003, 16 (1): 63-75. 10.1023/A:1020718718550.View ArticlePubMedGoogle Scholar
- Moos T: Immunohistochemical localization of intraneuronal transferrin receptor immunoreactivity in the adult mouse central nervous system. J Comp Neurol. 1996, 375 (4): 675-692. 10.1002/(SICI)1096-9861(19961125)375:4<675::AID-CNE8>3.0.CO;2-Z.View ArticlePubMedGoogle Scholar
- Leibold EA, Gahring LC, Rogers SW: Immunolocalization of iron regulatory protein expression in the murine central nervous system. Histochem Cell Biol. 2001, 115 (3): 195-203.PubMedGoogle Scholar
- Rodriguez A, Pan P, Parkkila S: Expression studies of neogenin and its ligand hemojuvelin in mouse tissues. J Histochem Cytochem. 2007, 55 (1): 85-96. 10.1369/jhc.6A7031.2006.View ArticlePubMedGoogle Scholar
- Zechel S, Huber-Wittmer K, von Bohlen O, Halbach O: Distribution of the iron-regulating protein hepcidin in the murine central nervous system. J Neurosci Res. 2006, 84 (4): 790-800. 10.1002/jnr.20991.View ArticlePubMedGoogle Scholar
- Wong RW, Richa DC, Hahn P, Green WR, Dunaief JL: Iron toxicity as a potential factor in AMD. Retina. 2007, 27 (8): 997-1003. 10.1097/IAE.0b013e318074c290.View ArticlePubMedGoogle Scholar
- Miwa H, Okawa M, Kajimoto Y, Kondo T: Transcranial sonography of the substantia nigra in patients with Parkinson's disease. J Neurol. 2007, 254 (Suppl 4): IV15-IV20. 10.1007/s00415-007-4004-z.Google Scholar
- Carri MT, Ferri A, Cozzolino M, Calabrese L, Rotilio G: Neurodegeneration in amyotrophic lateral sclerosis: the role of oxidative stress and altered homeostasis of metals. Brain Res Bull. 2003, 61 (4): 365-374. 10.1016/S0361-9230(03)00179-5.View ArticlePubMedGoogle Scholar
- Collingwood J, Dobson J: Mapping and characterization of iron compounds in Alzheimer's tissue. J Alzheimers Dis. 2006, 10 (2–3): 215-222.PubMedGoogle Scholar
- Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin. 2005, 55 (2): 74-108. 10.3322/canjclin.55.2.74.View ArticlePubMedGoogle Scholar
- Reardon DA, Rich JN, Friedman HS, Bigner DD: Recent advances in the treatment of malignant astrocytoma. J Clin Oncol. 2006, 24 (8): 1253-1265. 10.1200/JCO.2005.04.5302.View ArticlePubMedGoogle Scholar
- Fischer I, Gagner JP, Law M, Newcomb EW, Zagzag D: Angiogenesis in gliomas: biology and molecular pathophysiology. Brain Pathol. 2005, 15 (4): 297-310.View ArticlePubMedGoogle Scholar
- Park CH, Valore EV, Waring AJ, Ganz T: Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem. 2001, 276 (11): 7806-7810. 10.1074/jbc.M008922200.View ArticlePubMedGoogle Scholar
- Ganz T: Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood. 2003, 102 (3): 783-788. 10.1182/blood-2003-03-0672.View ArticlePubMedGoogle Scholar
- Atanasiu V, Manolescu B, Stoian I: Hepcidin – central regulator of iron metabolism. Eur J Haematol. 2007, 78 (1): 1-10. 10.1111/j.1600-0609.2006.00772.x.View ArticlePubMedGoogle Scholar
- Peyssonnaux C, Zinkernagel AS, Schuepbach RA, Rankin E, Vaulont S, Haase VH, Nizet V, Johnson RS: Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs). J Clin Invest. 2007, 117 (7): 1926-1932. 10.1172/JCI31370.PubMed CentralView ArticlePubMedGoogle Scholar
- Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J: Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004, 306 (5704): 2090-2093. 10.1126/science.1104742.View ArticlePubMedGoogle Scholar
- Zhang AS, West AP, Wyman AE, Bjorkman PJ, Enns CA: Interaction of hemojuvelin with neogenin results in iron accumulation in human embryonic kidney 293 cells. J Biol Chem. 2005, 280 (40): 33885-33894. 10.1074/jbc.M506207200.View ArticlePubMedGoogle Scholar
- Ganz T: Iron homeostasis: fitting the puzzle pieces together. Cell Metab. 2008, 7 (4): 288-290. 10.1016/j.cmet.2008.03.008.View ArticlePubMedGoogle Scholar
- Yang F, West AP, Allendorph GP, Choe S, Bjorkman PJ: Neogenin interacts with hemojuvelin through its two membrane-proximal fibronectin type III domains. Biochemistry. 2008, 47 (14): 4237-4245. 10.1021/bi800036h.PubMed CentralView ArticlePubMedGoogle Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3 (7): RESEARCH0034-10.1186/gb-2002-3-7-research0034.PubMed CentralView ArticlePubMedGoogle Scholar
- Bao B, Peatman E, Li P, He C, Liu Z: Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection. Dev Comp Immunol. 2005, 29 (11): 939-950. 10.1016/j.dci.2005.03.006.View ArticlePubMedGoogle Scholar
- Bertout JA, Patel SA, Simon MC: The impact of O2 availability on human cancer. Nat Rev Cancer. 2008, 8 (12): 967-975. 10.1038/nrc2540.PubMed CentralView ArticlePubMedGoogle Scholar
- Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S: The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest. 2002, 110 (7): 1037-1044.PubMed CentralView ArticlePubMedGoogle Scholar
- Lal A, Peters H, St Croix B, Haroon ZA, Dewhirst MW, Strausberg RL, Kaanders JH, Kogel van der AJ, Riggins GJ: Transcriptional response to hypoxia in human tumors. J Natl Cancer Inst. 2001, 93 (17): 1337-1343. 10.1093/jnci/93.17.1337.View ArticlePubMedGoogle Scholar
- Schofield CJ, Ratcliffe PJ: Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol. 2004, 5 (5): 343-354. 10.1038/nrm1366.View ArticlePubMedGoogle Scholar
- Silvestri L, Pagani A, Camaschella C: Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron homeostasis. Blood. 2008, 111 (2): 924-931. 10.1182/blood-2007-07-100677.View ArticlePubMedGoogle Scholar
- Zhang AS, Yang F, Meyer K, Hernandez C, Chapman-Arvedson T, Bjorkman PJ, Enns CA: Neogenin-mediated hemojuvelin shedding occurs after hemojuvelin traffics to the plasma membrane. J Biol Chem. 2008, 283 (25): 17494-17502. 10.1074/jbc.M710527200.PubMed CentralView ArticlePubMedGoogle Scholar
- Hu YC, Lam KY, Law S, Wong J, Srivastava G: Identification of differentially expressed genes in esophageal squamous cell carcinoma (ESCC) by cDNA expression array: overexpression of Fra-1, Neogenin, Id-1, and CDC25B genes in ESCC. Clin Cancer Res. 2001, 7 (8): 2213-2221.PubMedGoogle Scholar
- Lee JE, Kim HJ, Bae JY, Kim SW, Park JS, Shin HJ, Han W, Kang KS, Noh DY: Neogenin expression may be inversely correlated to the tumorigenicity of human breast cancer. BMC Cancer. 2005, 5: 154-10.1186/1471-2407-5-154.PubMed CentralView ArticlePubMedGoogle Scholar
- Recht L, Torres CO, Smith TW, Raso V, Griffin TW: Transferrin receptor in normal and neoplastic brain tissue: implications for brain-tumor immunotherapy. J Neurosurg. 1990, 72 (6): 941-945.View ArticlePubMedGoogle Scholar
- Fleming RE, Britton RS: Iron Imports. VI. HFE and regulation of intestinal iron absorption. Am J Physiol Gastrointest Liver Physiol. 2006, 290 (4): G590-594. 10.1152/ajpgi.00486.2005.View ArticlePubMedGoogle Scholar
- Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R, Ellis MC, Fullan A, et al.: A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996, 13 (4): 399-408. 10.1038/ng0896-399.View ArticlePubMedGoogle Scholar
- Guerreiro RJ, Bras JM, Santana I, Januario C, Santiago B, Morgadinho AS, Ribeiro MH, Hardy J, Singleton A, Oliveira C: Association of HFE common mutations with Parkinson's disease, Alzheimer's disease and mild cognitive impairment in a Portuguese cohort. BMC Neurol. 2006, 6: 24-10.1186/1471-2377-6-24.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang XS, Lee S, Simmons Z, Boyer P, Scott K, Liu W, Connor J: Increased incidence of the Hfe mutation in amyotrophic lateral sclerosis and related cellular consequences. J Neurol Sci. 2004, 227 (1): 27-33. 10.1016/j.jns.2004.08.003.View ArticlePubMedGoogle Scholar
- Connor JR, Lee SY: HFE mutations and Alzheimer's disease. J Alzheimers Dis. 2006, 10 (2–3): 267-276.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.