Neurochem Res DOI 10.1007/s11064-014-1285-3
ORIGINAL PAPER
Angiotensin II Inhibits Iron Uptake and Release in Cultured Neurons Yong Liu • Suna Huang • Fang Du • Guang Yang • Li Rong Jiang • Chao Zhang Zhong-ming Qian
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Received: 4 November 2013 / Revised: 14 February 2014 / Accepted: 13 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Based on the well-confirmed roles of angiotensin II (ANGII) in iron transport of peripheral organs and cells, the causative link of excess brain iron with and the involvement of ANGII in neurodegenerative disorders, we speculated that ANGII might also have an effect on expression of iron transport proteins in the brain. In the present study, we investigated effects of ANGII on iron uptake and release using the radio-isotope methods as well as expression of cell iron transport proteins by Western blot analysis in cultured neurons. Our findings demonstrated for the first time that ANGII significantly reduced transferrinbound iron and non-transferrin bound iron uptake and iron release as well as expression of two major iron uptake proteins transferrin receptor 1 and divalent metal transporter 1 and the key iron exporter ferroportin 1 in cultured neurons. The findings suggested that endogenous ANGII might have a physiological significance in brain iron metabolism. Keywords Renin–angiotensin system (RAS) Angiotensin II (ANGII) Neurons Transferrin-bound iron (Tf-Fe) Non-transferrin bound iron (NTBI) Iron release
Y. Liu Department of Neurology, Xin Qiao Hospital, Third Military Medical University, Chongqing 400038, China S. Huang G. Yang L. R. Jiang C. Zhang Z. Qian (&) Laboratory of Neuropharmacology and Department of Neurosurgery, South-West Hospital, Third Military Medical University, Chongqing 400038, China e-mail: [email protected] F. Du School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong
Abbreviations ACE AD ANGII AT1 and AT2 DMT1 - IRE DMT1 ? IRE Fpn1 GFAP iNOS MAP2 NTBI PBS PD RAS Tf-Fe TfR1
Angiotensin-converting enzyme Alzheimer’s disease Angiotensin II Angiotensin II (ANGII) type 1 receptor and type 2 receptor Divalent metal transporter 1 without iron response element Divalent metal transporter 1 with iron response element Ferroportin 1 Glial fibrillary acidic protein Inducible nitric oxide synthase Microtubule-associated protein 2 Non-transferrin bound iron Phosphate buffered saline Parkinson’s disease Renin–angiotensin system Transferrin-bound iron Transferrin receptor 1
Introduction The renin–angiotensin system (RAS) is best known for its role in regulating blood pressure, activation of sympathetic pathway, stimulation of vasopressin release, drinking behavior and cerebral blood flow [14]. The biological actions of the RAS are primarily mediated by the highly active octapeptide angiotensin II (ANGII) [6]. In addition to the systemic (circulating) RAS, there is evidence to indicate that many tissues, including the vasculature, heart, kidney and brain, are capable of producing ANGII, which
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may thereby mediate autocrine, paracrine and intracrine effects [3, 24]. Recently, studies have evidenced that ANGII has the function to regulate the expression of iron metabolism proteins and hence affect iron homeostasis in some peripheral organs and cells. Administration of ANGII has been demonstrated to cause iron accumulation and ferritin induction in the rat aorta [21], kidney, heart and liver [22]. It has also been reported that ANGII is able to induce a significant increase in the expression of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and hepcidin in the rat kidney [23]. In addition, ANGII has been found to promote non-transferrin-bound iron (NTBI) uptake by bovine endothelial cells [32] and facilitated the expression of iron uptake and release proteins including TfR1, DMT1 and Fpn1 and increased the intracellular iron concentration as well as labile ferrous iron in human glomerular endothelial cells [44]. ANGIIinduced deposition of iron has been considered to be at least partly associated with ANGII-induced renal or endothelial injury, impairment of vascular function, and arterial remodeling by the enhancement of oxidative stress [20]. In the mammalian brain, all the required components of the RAS, including renin, angiotensinogen, ACE, ANGII and its receptors are present [9, 19, 30, 34]. Also, all key proteins involved in iron metabolism and transport, such as TfR, DMT1, Fpn1 and iron regulatory peptide hepcidin, are expressed in the brain [27, 37, 38, 45, 47]. However, it is unknown whether ANGII could affect the expression of iron metabolism proteins and then iron homeostasis in the brain. Based on the well-confirmed functions of ANGII in peripheral iron metabolism, the causative link between excess brain iron and neurodegenerative disorders [25, 26], plus the recent studies on the role of ANGII in neurodegenerative diseases [13, 15, 28, 29, 31, 46], we speculated that ANGII might also have an effect on the expression of iron transport proteins in the brain. To test this hypothesis, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured neurons.
Materials and Methods Materials Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company, St. Louis, MO, USA. The scintillation cocktail and tubes were purchased from Beckman Coulter Company, Fullerton, CA, USA and 55FeCl3 from Perkinelmer Company, Wellesley, MA, USA. The mouse anti-rat TfR1 monoclonal antibody was obtained from BD
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Transduction Laboratories, BD Biosciences Pharmingen, USA and antibodies against DMT1 with (DMT1 ? IRE) or without iron response element (DMT1 - IRE) and Fpn1 were purchased from Alpha Diagnostic International Company, San Antonio, TX, USA. The specific antibodies against neuron microtubule-associated protein 2 (MAP2) and astrocyte glial fibrillary acidic protein (GFAP) were purchased from Chemicon International Ltd, UK. Male Sprague–Dawley (SD) rats were obtained from the animal center of the Third Military Medical University. The Animal Ethics Committees of the University approved the use of animals for this study. Primary Cortical Neurons Primary cortical neurons were prepared as previously described [18, 48], with minor modifications. In brief, the prefrontal cortex was aseptically removed from 1-day-old SD rats, minced with sterile surgical blades, incubated in 0.125 % trypsin and dissociated by trituration in DNase/ trypsin inhibitor solution. Dissociated cortical cells were diluted in DMEM/Ham’s F12 medium (1:1, v/v, pH 7.2) containing 10 % heart-inactivated FBS glutamine (4 mM), 4.5 g/l D-glucose and penicillin (100 U), and then plated on poly L-lysine-coated coverslips. Non-neuronal cell division was inhibited by exposure to cytarabine (Sigma) for 24-h. The cultures were maintained at 37 °C in a humidified environment with 5 % CO2 in a CO2 incubator (TC2323). The purity of these cultures was assessed by staining for the neuron specific antibodies against MAP2 and the astrocyte marker GFAP and reached approximately 98 %. Measurement of Transferrin-Bound Iron (Tf-Fe) Uptake 55
Fe-Tf was prepared first by mixing 55FeCl3 (PerkinElmer) with nitrilotriacetic acid (NTA) in a 1:10 ratio and then incubating 55Fe-NTA with apo-Tf in a 2:1 ratio for 3-h in carbonate buffer (10 mM NaHCO3, 250 mM Tris–HCl) [35, 36]. After being incubated with serum-free culture medium containing 0.1 % BSA at 37 °C for 1-h to remove any endogenous transferrin, the cells were treated with ANGII for the designed periods and then incubated with or without 55Fe-Tf for 30-min. After centrifugation to remove cell surface-bound radioactive 55Fe-Tf, the cells were washed again with ice-cold phosphate buffered saline (PBS), lysed in 1 % SDS, and counted for 10-min in a scintillation counter (Perkin-Elmer). The counts represented the55Fe taken up by the cells and was then calculated by converting cellular radioactivity in cpm to a value of iron concentration in pmol according to the iron standard curve and divided by the protein amount, and presented as
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pmol/lg protein. The protein content was determined using the Bradford assay kit (Bio-Rad). Measurement of Non-transferrin Bound Iron Uptake The radio-labelled 55Fe(II) (NTBI) solution was prepared and the Fe(II) uptake measured as described previously [39]. After being incubated with ANGII for the designed periods and then 55Fe(II) for 30-min, the cells were lysed, scraped off and transferred into Eppendorf tubes. A 50 ll aliquot was subjected to the detection of protein concentration. The cytosol was separated from the stromal fraction by centrifugation at 10,000g for 20-min at 4 °C using a Jouan centrifuge (DJB labcare Ltd., England). Scintillation solution (3 ml) was added to determine the counts per minute (cpm). The total iron uptake was the sum of the cytosol and stromal fractions.
fered saline containing 0.1 % Tween-20 at 4 °C overnight. The membrane was rinsed in three changes of Tris-buffered saline/Tween-20, incubated in fresh washing buffer once for 15-min and twice for 5-min, and then incubated overnight at 4 °C with primary antibodies: mouse anti-rat TfR1 monoclonal antibody (1:1,000); rabbit anti-rat DMT1 ? IRE, DMT1 - IRE polyclonal antibodies and rabbit anti-mouse Fpn1 polyclonal antibody (1:5,000). After three washes, the blots were incubated with goat antirabbit or anti-mouse IRDye 800 CW secondary antibody (1:5,000, Li-Cor) for 1-h at room temperature. The intensity of the specific bands was detected and analyzed by Odyssey infrared imagine system (Li-Cor). To ensure even loading of the samples, the same membrane was probed with rabbit anti-rat b-actin polyclonal antibody at a 1:2,000 dilution [4]. Statistical Analysis
Iron Release Assay The neurons were incubated with 55Fe(II) and then with ANGII. The medium was then collected and the cpm measured following centrifugation. The cells were detached by 500 ll lysis buffer. A 50-ll aliquot was subjected to the detection of the protein concentration. The cytosol was separated from the stromal fraction by centrifugation at 10,000g at 4 °C for 20-min. A 3 ml scintillation solution was added to both fractions to count the cpm. The sum of the radioactivity in the medium and in the cell (cytosol and stromal fractions) was named the total cellular radioactivity. The relative percentage of the total radioactivity in the medium and in the cell was calculated. The percentage of 55Fe release was calculated according to the following equation: % 55Fe release = [(cpm in supernatant)/(cpm in supernatant ? cpm in cells)] 9 100 [11]. Western Blot Analysis Neurons received different treatments were washed with ice-cold PBS, homogenized with lysis buffer and then subjected to sonication using Soniprep 150 (MSE Scientific Instruments, London, UK). After centrifugation at 10,000g and at 4 °C for 15-min, the supernatant was collected, and protein content was determined using the BioRad. Aliquots of the cell extract containing 30 lg of protein were diluted in 2 ml sample buffer (50 mM Tris, pH 6.8, 2 % SDS, 10 % glycerol, 0.1 % bromphenol blue, and 5 % b-mercaptoethanol) and heated for 5-min at 95 °C before SDS-PAGE on 10 % gel and subsequently transferred to a pure nitrocellulose membrane. The membrane was then blocked with 5 % blocking reagent in Tris-buf-
Statistical analyses was performed using SPSS software for Windows (version 10.0) (SPSS, Inc., Chicago, IL, USA). Data were presented as mean ± SEM. The difference between the means was determined by one-way ANOVA followed by a Student–Newman–Keuls test for multiple comparisons. A probability value of P \ 0.05 was taken to be statistically significant. Correlations were analyzed by Pearson’s bivariate correlations using SPSS.
Results Angiotensin II Inhibited Transferrin-Bound Iron and Non-transferrin Bound Iron Uptake by Cultured Neurons To determine the effects of ANGII on Tf-Fe and NTBI uptake by neurons, the cells were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C and then incubated with 55Fe(II) (1 lM) or 55Fe-Tf (2 lM) for 30-min. In our preliminary study on effects of different concentrations of ANGII on iron contents, expression of iron transport proteins and cell viability, it was found that 100 nM of ANGII induced a peak reaction in iron contents and expression of iron transport proteins, and had no effect on cell viability, thus this concentration was used in this study. After the treatments, Tf-Fe and NTBI uptake by the cells was assayed. The radio-isotope measurements showed that treatment of neurons with ANGII induced a remarkable decrease in Tf-Fe and NTBI uptake. The amounts of Tf-Fe (Fig. 2a) uptake in the neurons treated with 100 nM for 6, 12 or 24-h were 29.557 ± 7.959, 26.856 ± 8.006 and 26.789 ± 6.971 pmol/lg protein; respectively. All of these
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values were significantly lower than the corresponding value (54.032 ± 8.939 pmol/lg protein) in the control neurons (P \ 0.05 or 0.01). There were no significant differences between Tf-Fe uptake by neurons treated with ANGII 100 nM for 3-h (49.337 ± 10.65) and the control neurons. The similar effects of ANGII were also found on NTBI uptake by neurons. The amounts of NTBI (Fig. 2b) uptake in the neurons treated with 100 nM for 6, 12 or 24-h were 39.112 ± 12.408, 32.021 ± 6.353 and 29.443 ± 3.321 pmol/lg protein; respectively. Also, all of these values were significantly lower than that (77.112 ± 9.715 pmol/lg protein) in the control cells (P \ 0.05 or 0.01). Again, no significant differences in NTBI uptake were found between neurons treated with ANGII 100 nM for 3-h (68.614 ± 11.841 pmol/lg protein) and the controls. Angiotensin II Inhibited Iron Release from the Cultured Neurons We then examined the effects of ANGII on iron release from neurons by incubation of cells with 55Fe(II) (2 lM) at 37 °C for 30-min and then with or without 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C. The cpm in the medium and in cells was measured respectively. The percentage of 55Fe release at 6, 12 or 24-h was 42.7491 ± 6.468, 57.331 ± 5.683 or 71.169 ± 2.631 in the neurons treated with 100 nM of ANGII, while the corresponding value in the control neurons at 6, 12 or 24-h was 58.4039 ± 5.261, 71.8576 ± 4.763 or 81.832 ± 3.3539; respectively. The significant differences were found at all of these time points, indicating that ANGII could significantly inhibit iron release from the cultured neurons.
Fig. 1 ANGII inhibited Tf-Fe and NTBI uptake and iron release in cultured neurons. Neurons were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C and then incubated with 55Fe(II) (1 lM) or 55 Fe-Tf (10 lg/ml) for 30-min. To examine the effects of ANGII on iron release from neurons, the cells were incubated 55Fe(II) (2 lM) at
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Angiotensin II Suppressed Expression of the Cellular Iron Uptake Proteins Transferrin Receptor 1 and Divalent Metal Transporter 1 in Neurons To understand why ANGII can inhibit Tf-Fe and NTBI uptake by neurons, we investigated effects of ANGII on the expression of iron uptake proteins (TfR1, DMT1 ? IRE and DMT1 - IRE) by pre-treatment of neurons with different concentrations of ANGII (0, 10 or 100 nM) for 0, 6, 12 or 24 h at 37 °C and the expression of these proteins was then determined by western blot analysis. It was found that ANGII resulted in a significant decrease in the expression of TfR1 (10 nM for 12 or 24-h, and 100 nM for 6, 12 or 24-h, P \ 0.05 or 0.01 vs. the control) (Fig. 2a), DMT1 ? IRE (10 or 100 nM for 6, 12 or 24-h, P \ 0.05 or 0.01 vs. the control) (Fig. 1b), DMT1 - IRE (10 nM for 12 or 24-h, and 100 nM for 6, 12 or 24-h, P \ 0.05 or 0.01 vs. the control) (Fig. 1c). This implied that the inhibitory role of ANGII on Tf-Fe and NTBI uptake is due to the reduced expression of major iron uptake proteins TfR1 and DMT1. Angiotensin II Suppressed Expression of the Cellular Iron Release Protein Ferroportin 1 in Neurons Fpn 1, also known as metal transport protein 1 (MTP1), ironregulated transporter 1 (IREG1), or Slc11a3, functions as the only known iron exporter in mammalian cells and plays a critical role in the maintenance of both cellular and systemic iron balance [7]. To explore the possible mechanisms of inhibition of ANGII on iron release from neurons, we also investigated effects of ANGII on the expression of iron release protein Fpn1 by pre-treatment of neurons with different concentrations of ANGII (0, 10 or 100 nM) for 0, 6, 12
37 °C for 30-min before treatment with or without 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C. After the treatments, Tf-Fe (a) and NTBI (b) uptake and iron release (c) in neurons were assayed as described in ‘‘Materials and Methods’’. All data were presented as mean ± SEM (n = 7). *P \ 0.05, **P \ 0.01 versus the control
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Fig. 2 ANGII suppressed expression of the cellular iron uptake and release proteins TfR1, DMT1 and Fpn1 in neurons. Neurons were treated with 0, 10 or 100 nM of ANGII for 0, 6 (dot), 12 (strip) or 24-h (solid) at 37 °C and the expression of iron uptake (TfR1,
DMT1 ? IRE and DMT1 - IRE) and release (Fpn1) proteins was then determined by western blot analysis. a TfR1, b DMT1 ? IRE, c DMT1 - IRE, and d Fpn1. All data were presented as mean ± SEM (n = 7). *P \ 0.05, **P \ 0.01 versus the control
or 24 h at 37 °C. Western blot analysis of Fpn1 expression demonstrated that ANGII could induce in a significant decrease in the expression of Fpn1 (Fig. 2d). The contents of Fpn1 in neurons treated with 10 nM for 12 or 24-h or 100 nM for 6, 12 or 24-h were significantly lower than those in the control cells (P \ 0.05 or 0.01). The findings implied that the significant inhibitory role of ANGII on iron release is associated with by its inhibitory effect on the expression of iron release protein Fpn1.
transport in neurons. Our findings demonstrated for the first time that ANGII has a significant negative effect on Tf-Fe and NTBI uptake, iron release, expression of two major iron uptake proteins TfR1 and DMT1 and the key iron exporter Fpn1 in cultured neurons. Iron is an essential element that is required for fundamental cell functions in all living cells including neurons [38, 40]. On the other hand, excess body iron is potentially harmful because of its ability to catalyze the conversion of hydrogen peroxide to toxic free radicals. Excess iron in the brain has been widely accepted as a primary cause of neurodegenerative disorders [25, 26]. Therefore, the full understanding of homeostatic mechanisms involved in brain iron metabolism is fundamental and critical for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain. The
Discussion To our knowledge, the current study is the first investigation on the relationship between ANGII or RAS and iron
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findings from the present study suggested that endogenous ANGII might have a physiological significance in brain iron homeostasis. A detailed investigation on the issue will make contribution to better understand how iron homeostasis in the brain is maintained. Recent findings from clinical trials and experimental studies have indicated that the brain-RAS may be associated with neurodegenerative diseases such as Alzheimer’s disease (AD), multiple sclerosis and Parkinson’s disease (PD) [5, 10, 46]. Examination of human postmortem brain tissues has shown a loss of both ANGII type 1 receptor (AT1) and type 2 receptor (AT2) binding sites in the substantia nigra of PD patients. This implies that PD-induced neurodegeneration may involve AT receptor expressing cells [10]. In addition, it has been demonstrated that that ANGII reduces the toxicity of alpha-synuclein in a genetic in vitro PD model produced by alpha-synuclein over-expression in the human neuroglioma H4 cell line [15]. ANGII is also reported to be able to protect primary E15 rat ventral mesencephalic (VM) dopamine neurons against the mitochondrial complex I inhibitor rotenone by reducing rotenone-induced caspase-3 activation and inhibiting subsequent apoptosis, and to protects dopaminergic neurons against 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary VM cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice [13, 14]. Based on the understanding of the relationship of iron with alpha-synuclein [2, 42], rotenone [33, 36] and MPTP [16], it is highly likely that the neuro-protective role of ANGII might be partly associated with its negative effect on iron uptake we found in the present study. In peripheral organs and cells, a number of studies have demonstrated that ANGII plays a positive role, rather than a negative effect as we found in neurons, in the regulation of the expression of iron metabolism proteins and the intracellular iron levels. It has been reported that ANGII administration is able to induce a significant increase in the expression of iron uptake and release proteins TfR1, DMT1 and Fpn1 and iron regulatory protein hepcidin in the rat kidney [23] and in human glomerular endothelial cells [44]. It has also been confirmed that treatment with ANGII could cause iron accumulation or the intracellular iron concentration, labile ferrous iron as well as ferritin induction in the rat aorta [21], kidney, heart and liver [22], and to promote NTBI uptake by bovine endothelial cells [32] and human glomerular endothelial cells [44]. The positive effect of ANGII on iron metabolism has been considered to be one of causes for ANGII-induced renal or endothelial injury, impairment of vascular function, and arterial remodeling [20]. These findings plus the results obtained from the current study indicated that there might be a regional or cellular specific regulation of ANGII on iron metabolism although the causes are unknown currently.
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The concentration of iron in the brain is region-dependent, and in the basal ganglia iron is present at a concentration equals to that found in the liver [1]. At the cell level, the oligodendrocyte is the predominant cell type that contains iron in the brain [41]. As in all cells, neurons require iron for many aspects of their physiology, including electron transport, NADPH reductase activity, myelination of axons, and as a cofactor for several enzymes involved in neurotransmitter synthesis. Hence, an imbalance in brain iron results in dysfunction in iron related metabolism [12, 43]. The regulation or management of iron at the cellular level is primarily by four proteins: TfR1, DMT1, Fpn1 and ferritin. By controlling their levels, the cell can determine the amount of iron acquired (proportional to the number of membrane TfR1 and DMT1), released (proportional to the number of membrane Fpn1) and sequestered (proportional to the cytoplasmic level of ferritin). In most types of cells, the coordinated control of TfR1, DMT1, Fpn1 and ferritin by cellular iron occurs at the post-transcriptional level and is mediated by cytoplasmic RNA binding proteins, known as the iron regulatory proteins (IRPs) [8, 17, 26], and also by the amount of a newly discovered peptide hepcidin. Currently, it is unknown whether there is a regional or cellular specific regulation of ANGII on iron transport in the brain. Also, a detailed analysis is needed to determine whether endogenous ANGII has a physiological significance or importance in brain iron metabolism. These studies are necessary for fully understanding of the mechanisms involved in bran iron homeostasis. Acknowledgments The studies in our laboratories were supported by the Competitive Earmarked Grants of The Hong Kong Research Grants Council (GRF 466713), National 973 Programs (2011CB510004), the General Grant of National Natural Science Foundation of China (NSFC) (31271132-2012, 31371092-2013), Key Project Grant of NSFC (31330035-2013) and Direct Grant of the Chinese University of Hong Kong (2011.1.084). Conflict of interest
The authors declare no conflict of interest.
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ORIGINAL PAPER
Angiotensin II Inhibits Iron Uptake and Release in Cultured Neurons Yong Liu • Suna Huang • Fang Du • Guang Yang • Li Rong Jiang • Chao Zhang Zhong-ming Qian
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Received: 4 November 2013 / Revised: 14 February 2014 / Accepted: 13 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Based on the well-confirmed roles of angiotensin II (ANGII) in iron transport of peripheral organs and cells, the causative link of excess brain iron with and the involvement of ANGII in neurodegenerative disorders, we speculated that ANGII might also have an effect on expression of iron transport proteins in the brain. In the present study, we investigated effects of ANGII on iron uptake and release using the radio-isotope methods as well as expression of cell iron transport proteins by Western blot analysis in cultured neurons. Our findings demonstrated for the first time that ANGII significantly reduced transferrinbound iron and non-transferrin bound iron uptake and iron release as well as expression of two major iron uptake proteins transferrin receptor 1 and divalent metal transporter 1 and the key iron exporter ferroportin 1 in cultured neurons. The findings suggested that endogenous ANGII might have a physiological significance in brain iron metabolism. Keywords Renin–angiotensin system (RAS) Angiotensin II (ANGII) Neurons Transferrin-bound iron (Tf-Fe) Non-transferrin bound iron (NTBI) Iron release
Y. Liu Department of Neurology, Xin Qiao Hospital, Third Military Medical University, Chongqing 400038, China S. Huang G. Yang L. R. Jiang C. Zhang Z. Qian (&) Laboratory of Neuropharmacology and Department of Neurosurgery, South-West Hospital, Third Military Medical University, Chongqing 400038, China e-mail: [email protected] F. Du School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong
Abbreviations ACE AD ANGII AT1 and AT2 DMT1 - IRE DMT1 ? IRE Fpn1 GFAP iNOS MAP2 NTBI PBS PD RAS Tf-Fe TfR1
Angiotensin-converting enzyme Alzheimer’s disease Angiotensin II Angiotensin II (ANGII) type 1 receptor and type 2 receptor Divalent metal transporter 1 without iron response element Divalent metal transporter 1 with iron response element Ferroportin 1 Glial fibrillary acidic protein Inducible nitric oxide synthase Microtubule-associated protein 2 Non-transferrin bound iron Phosphate buffered saline Parkinson’s disease Renin–angiotensin system Transferrin-bound iron Transferrin receptor 1
Introduction The renin–angiotensin system (RAS) is best known for its role in regulating blood pressure, activation of sympathetic pathway, stimulation of vasopressin release, drinking behavior and cerebral blood flow [14]. The biological actions of the RAS are primarily mediated by the highly active octapeptide angiotensin II (ANGII) [6]. In addition to the systemic (circulating) RAS, there is evidence to indicate that many tissues, including the vasculature, heart, kidney and brain, are capable of producing ANGII, which
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may thereby mediate autocrine, paracrine and intracrine effects [3, 24]. Recently, studies have evidenced that ANGII has the function to regulate the expression of iron metabolism proteins and hence affect iron homeostasis in some peripheral organs and cells. Administration of ANGII has been demonstrated to cause iron accumulation and ferritin induction in the rat aorta [21], kidney, heart and liver [22]. It has also been reported that ANGII is able to induce a significant increase in the expression of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and hepcidin in the rat kidney [23]. In addition, ANGII has been found to promote non-transferrin-bound iron (NTBI) uptake by bovine endothelial cells [32] and facilitated the expression of iron uptake and release proteins including TfR1, DMT1 and Fpn1 and increased the intracellular iron concentration as well as labile ferrous iron in human glomerular endothelial cells [44]. ANGIIinduced deposition of iron has been considered to be at least partly associated with ANGII-induced renal or endothelial injury, impairment of vascular function, and arterial remodeling by the enhancement of oxidative stress [20]. In the mammalian brain, all the required components of the RAS, including renin, angiotensinogen, ACE, ANGII and its receptors are present [9, 19, 30, 34]. Also, all key proteins involved in iron metabolism and transport, such as TfR, DMT1, Fpn1 and iron regulatory peptide hepcidin, are expressed in the brain [27, 37, 38, 45, 47]. However, it is unknown whether ANGII could affect the expression of iron metabolism proteins and then iron homeostasis in the brain. Based on the well-confirmed functions of ANGII in peripheral iron metabolism, the causative link between excess brain iron and neurodegenerative disorders [25, 26], plus the recent studies on the role of ANGII in neurodegenerative diseases [13, 15, 28, 29, 31, 46], we speculated that ANGII might also have an effect on the expression of iron transport proteins in the brain. To test this hypothesis, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured neurons.
Materials and Methods Materials Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company, St. Louis, MO, USA. The scintillation cocktail and tubes were purchased from Beckman Coulter Company, Fullerton, CA, USA and 55FeCl3 from Perkinelmer Company, Wellesley, MA, USA. The mouse anti-rat TfR1 monoclonal antibody was obtained from BD
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Transduction Laboratories, BD Biosciences Pharmingen, USA and antibodies against DMT1 with (DMT1 ? IRE) or without iron response element (DMT1 - IRE) and Fpn1 were purchased from Alpha Diagnostic International Company, San Antonio, TX, USA. The specific antibodies against neuron microtubule-associated protein 2 (MAP2) and astrocyte glial fibrillary acidic protein (GFAP) were purchased from Chemicon International Ltd, UK. Male Sprague–Dawley (SD) rats were obtained from the animal center of the Third Military Medical University. The Animal Ethics Committees of the University approved the use of animals for this study. Primary Cortical Neurons Primary cortical neurons were prepared as previously described [18, 48], with minor modifications. In brief, the prefrontal cortex was aseptically removed from 1-day-old SD rats, minced with sterile surgical blades, incubated in 0.125 % trypsin and dissociated by trituration in DNase/ trypsin inhibitor solution. Dissociated cortical cells were diluted in DMEM/Ham’s F12 medium (1:1, v/v, pH 7.2) containing 10 % heart-inactivated FBS glutamine (4 mM), 4.5 g/l D-glucose and penicillin (100 U), and then plated on poly L-lysine-coated coverslips. Non-neuronal cell division was inhibited by exposure to cytarabine (Sigma) for 24-h. The cultures were maintained at 37 °C in a humidified environment with 5 % CO2 in a CO2 incubator (TC2323). The purity of these cultures was assessed by staining for the neuron specific antibodies against MAP2 and the astrocyte marker GFAP and reached approximately 98 %. Measurement of Transferrin-Bound Iron (Tf-Fe) Uptake 55
Fe-Tf was prepared first by mixing 55FeCl3 (PerkinElmer) with nitrilotriacetic acid (NTA) in a 1:10 ratio and then incubating 55Fe-NTA with apo-Tf in a 2:1 ratio for 3-h in carbonate buffer (10 mM NaHCO3, 250 mM Tris–HCl) [35, 36]. After being incubated with serum-free culture medium containing 0.1 % BSA at 37 °C for 1-h to remove any endogenous transferrin, the cells were treated with ANGII for the designed periods and then incubated with or without 55Fe-Tf for 30-min. After centrifugation to remove cell surface-bound radioactive 55Fe-Tf, the cells were washed again with ice-cold phosphate buffered saline (PBS), lysed in 1 % SDS, and counted for 10-min in a scintillation counter (Perkin-Elmer). The counts represented the55Fe taken up by the cells and was then calculated by converting cellular radioactivity in cpm to a value of iron concentration in pmol according to the iron standard curve and divided by the protein amount, and presented as
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pmol/lg protein. The protein content was determined using the Bradford assay kit (Bio-Rad). Measurement of Non-transferrin Bound Iron Uptake The radio-labelled 55Fe(II) (NTBI) solution was prepared and the Fe(II) uptake measured as described previously [39]. After being incubated with ANGII for the designed periods and then 55Fe(II) for 30-min, the cells were lysed, scraped off and transferred into Eppendorf tubes. A 50 ll aliquot was subjected to the detection of protein concentration. The cytosol was separated from the stromal fraction by centrifugation at 10,000g for 20-min at 4 °C using a Jouan centrifuge (DJB labcare Ltd., England). Scintillation solution (3 ml) was added to determine the counts per minute (cpm). The total iron uptake was the sum of the cytosol and stromal fractions.
fered saline containing 0.1 % Tween-20 at 4 °C overnight. The membrane was rinsed in three changes of Tris-buffered saline/Tween-20, incubated in fresh washing buffer once for 15-min and twice for 5-min, and then incubated overnight at 4 °C with primary antibodies: mouse anti-rat TfR1 monoclonal antibody (1:1,000); rabbit anti-rat DMT1 ? IRE, DMT1 - IRE polyclonal antibodies and rabbit anti-mouse Fpn1 polyclonal antibody (1:5,000). After three washes, the blots were incubated with goat antirabbit or anti-mouse IRDye 800 CW secondary antibody (1:5,000, Li-Cor) for 1-h at room temperature. The intensity of the specific bands was detected and analyzed by Odyssey infrared imagine system (Li-Cor). To ensure even loading of the samples, the same membrane was probed with rabbit anti-rat b-actin polyclonal antibody at a 1:2,000 dilution [4]. Statistical Analysis
Iron Release Assay The neurons were incubated with 55Fe(II) and then with ANGII. The medium was then collected and the cpm measured following centrifugation. The cells were detached by 500 ll lysis buffer. A 50-ll aliquot was subjected to the detection of the protein concentration. The cytosol was separated from the stromal fraction by centrifugation at 10,000g at 4 °C for 20-min. A 3 ml scintillation solution was added to both fractions to count the cpm. The sum of the radioactivity in the medium and in the cell (cytosol and stromal fractions) was named the total cellular radioactivity. The relative percentage of the total radioactivity in the medium and in the cell was calculated. The percentage of 55Fe release was calculated according to the following equation: % 55Fe release = [(cpm in supernatant)/(cpm in supernatant ? cpm in cells)] 9 100 [11]. Western Blot Analysis Neurons received different treatments were washed with ice-cold PBS, homogenized with lysis buffer and then subjected to sonication using Soniprep 150 (MSE Scientific Instruments, London, UK). After centrifugation at 10,000g and at 4 °C for 15-min, the supernatant was collected, and protein content was determined using the BioRad. Aliquots of the cell extract containing 30 lg of protein were diluted in 2 ml sample buffer (50 mM Tris, pH 6.8, 2 % SDS, 10 % glycerol, 0.1 % bromphenol blue, and 5 % b-mercaptoethanol) and heated for 5-min at 95 °C before SDS-PAGE on 10 % gel and subsequently transferred to a pure nitrocellulose membrane. The membrane was then blocked with 5 % blocking reagent in Tris-buf-
Statistical analyses was performed using SPSS software for Windows (version 10.0) (SPSS, Inc., Chicago, IL, USA). Data were presented as mean ± SEM. The difference between the means was determined by one-way ANOVA followed by a Student–Newman–Keuls test for multiple comparisons. A probability value of P \ 0.05 was taken to be statistically significant. Correlations were analyzed by Pearson’s bivariate correlations using SPSS.
Results Angiotensin II Inhibited Transferrin-Bound Iron and Non-transferrin Bound Iron Uptake by Cultured Neurons To determine the effects of ANGII on Tf-Fe and NTBI uptake by neurons, the cells were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C and then incubated with 55Fe(II) (1 lM) or 55Fe-Tf (2 lM) for 30-min. In our preliminary study on effects of different concentrations of ANGII on iron contents, expression of iron transport proteins and cell viability, it was found that 100 nM of ANGII induced a peak reaction in iron contents and expression of iron transport proteins, and had no effect on cell viability, thus this concentration was used in this study. After the treatments, Tf-Fe and NTBI uptake by the cells was assayed. The radio-isotope measurements showed that treatment of neurons with ANGII induced a remarkable decrease in Tf-Fe and NTBI uptake. The amounts of Tf-Fe (Fig. 2a) uptake in the neurons treated with 100 nM for 6, 12 or 24-h were 29.557 ± 7.959, 26.856 ± 8.006 and 26.789 ± 6.971 pmol/lg protein; respectively. All of these
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values were significantly lower than the corresponding value (54.032 ± 8.939 pmol/lg protein) in the control neurons (P \ 0.05 or 0.01). There were no significant differences between Tf-Fe uptake by neurons treated with ANGII 100 nM for 3-h (49.337 ± 10.65) and the control neurons. The similar effects of ANGII were also found on NTBI uptake by neurons. The amounts of NTBI (Fig. 2b) uptake in the neurons treated with 100 nM for 6, 12 or 24-h were 39.112 ± 12.408, 32.021 ± 6.353 and 29.443 ± 3.321 pmol/lg protein; respectively. Also, all of these values were significantly lower than that (77.112 ± 9.715 pmol/lg protein) in the control cells (P \ 0.05 or 0.01). Again, no significant differences in NTBI uptake were found between neurons treated with ANGII 100 nM for 3-h (68.614 ± 11.841 pmol/lg protein) and the controls. Angiotensin II Inhibited Iron Release from the Cultured Neurons We then examined the effects of ANGII on iron release from neurons by incubation of cells with 55Fe(II) (2 lM) at 37 °C for 30-min and then with or without 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C. The cpm in the medium and in cells was measured respectively. The percentage of 55Fe release at 6, 12 or 24-h was 42.7491 ± 6.468, 57.331 ± 5.683 or 71.169 ± 2.631 in the neurons treated with 100 nM of ANGII, while the corresponding value in the control neurons at 6, 12 or 24-h was 58.4039 ± 5.261, 71.8576 ± 4.763 or 81.832 ± 3.3539; respectively. The significant differences were found at all of these time points, indicating that ANGII could significantly inhibit iron release from the cultured neurons.
Fig. 1 ANGII inhibited Tf-Fe and NTBI uptake and iron release in cultured neurons. Neurons were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C and then incubated with 55Fe(II) (1 lM) or 55 Fe-Tf (10 lg/ml) for 30-min. To examine the effects of ANGII on iron release from neurons, the cells were incubated 55Fe(II) (2 lM) at
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Angiotensin II Suppressed Expression of the Cellular Iron Uptake Proteins Transferrin Receptor 1 and Divalent Metal Transporter 1 in Neurons To understand why ANGII can inhibit Tf-Fe and NTBI uptake by neurons, we investigated effects of ANGII on the expression of iron uptake proteins (TfR1, DMT1 ? IRE and DMT1 - IRE) by pre-treatment of neurons with different concentrations of ANGII (0, 10 or 100 nM) for 0, 6, 12 or 24 h at 37 °C and the expression of these proteins was then determined by western blot analysis. It was found that ANGII resulted in a significant decrease in the expression of TfR1 (10 nM for 12 or 24-h, and 100 nM for 6, 12 or 24-h, P \ 0.05 or 0.01 vs. the control) (Fig. 2a), DMT1 ? IRE (10 or 100 nM for 6, 12 or 24-h, P \ 0.05 or 0.01 vs. the control) (Fig. 1b), DMT1 - IRE (10 nM for 12 or 24-h, and 100 nM for 6, 12 or 24-h, P \ 0.05 or 0.01 vs. the control) (Fig. 1c). This implied that the inhibitory role of ANGII on Tf-Fe and NTBI uptake is due to the reduced expression of major iron uptake proteins TfR1 and DMT1. Angiotensin II Suppressed Expression of the Cellular Iron Release Protein Ferroportin 1 in Neurons Fpn 1, also known as metal transport protein 1 (MTP1), ironregulated transporter 1 (IREG1), or Slc11a3, functions as the only known iron exporter in mammalian cells and plays a critical role in the maintenance of both cellular and systemic iron balance [7]. To explore the possible mechanisms of inhibition of ANGII on iron release from neurons, we also investigated effects of ANGII on the expression of iron release protein Fpn1 by pre-treatment of neurons with different concentrations of ANGII (0, 10 or 100 nM) for 0, 6, 12
37 °C for 30-min before treatment with or without 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C. After the treatments, Tf-Fe (a) and NTBI (b) uptake and iron release (c) in neurons were assayed as described in ‘‘Materials and Methods’’. All data were presented as mean ± SEM (n = 7). *P \ 0.05, **P \ 0.01 versus the control
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Fig. 2 ANGII suppressed expression of the cellular iron uptake and release proteins TfR1, DMT1 and Fpn1 in neurons. Neurons were treated with 0, 10 or 100 nM of ANGII for 0, 6 (dot), 12 (strip) or 24-h (solid) at 37 °C and the expression of iron uptake (TfR1,
DMT1 ? IRE and DMT1 - IRE) and release (Fpn1) proteins was then determined by western blot analysis. a TfR1, b DMT1 ? IRE, c DMT1 - IRE, and d Fpn1. All data were presented as mean ± SEM (n = 7). *P \ 0.05, **P \ 0.01 versus the control
or 24 h at 37 °C. Western blot analysis of Fpn1 expression demonstrated that ANGII could induce in a significant decrease in the expression of Fpn1 (Fig. 2d). The contents of Fpn1 in neurons treated with 10 nM for 12 or 24-h or 100 nM for 6, 12 or 24-h were significantly lower than those in the control cells (P \ 0.05 or 0.01). The findings implied that the significant inhibitory role of ANGII on iron release is associated with by its inhibitory effect on the expression of iron release protein Fpn1.
transport in neurons. Our findings demonstrated for the first time that ANGII has a significant negative effect on Tf-Fe and NTBI uptake, iron release, expression of two major iron uptake proteins TfR1 and DMT1 and the key iron exporter Fpn1 in cultured neurons. Iron is an essential element that is required for fundamental cell functions in all living cells including neurons [38, 40]. On the other hand, excess body iron is potentially harmful because of its ability to catalyze the conversion of hydrogen peroxide to toxic free radicals. Excess iron in the brain has been widely accepted as a primary cause of neurodegenerative disorders [25, 26]. Therefore, the full understanding of homeostatic mechanisms involved in brain iron metabolism is fundamental and critical for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain. The
Discussion To our knowledge, the current study is the first investigation on the relationship between ANGII or RAS and iron
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findings from the present study suggested that endogenous ANGII might have a physiological significance in brain iron homeostasis. A detailed investigation on the issue will make contribution to better understand how iron homeostasis in the brain is maintained. Recent findings from clinical trials and experimental studies have indicated that the brain-RAS may be associated with neurodegenerative diseases such as Alzheimer’s disease (AD), multiple sclerosis and Parkinson’s disease (PD) [5, 10, 46]. Examination of human postmortem brain tissues has shown a loss of both ANGII type 1 receptor (AT1) and type 2 receptor (AT2) binding sites in the substantia nigra of PD patients. This implies that PD-induced neurodegeneration may involve AT receptor expressing cells [10]. In addition, it has been demonstrated that that ANGII reduces the toxicity of alpha-synuclein in a genetic in vitro PD model produced by alpha-synuclein over-expression in the human neuroglioma H4 cell line [15]. ANGII is also reported to be able to protect primary E15 rat ventral mesencephalic (VM) dopamine neurons against the mitochondrial complex I inhibitor rotenone by reducing rotenone-induced caspase-3 activation and inhibiting subsequent apoptosis, and to protects dopaminergic neurons against 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary VM cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice [13, 14]. Based on the understanding of the relationship of iron with alpha-synuclein [2, 42], rotenone [33, 36] and MPTP [16], it is highly likely that the neuro-protective role of ANGII might be partly associated with its negative effect on iron uptake we found in the present study. In peripheral organs and cells, a number of studies have demonstrated that ANGII plays a positive role, rather than a negative effect as we found in neurons, in the regulation of the expression of iron metabolism proteins and the intracellular iron levels. It has been reported that ANGII administration is able to induce a significant increase in the expression of iron uptake and release proteins TfR1, DMT1 and Fpn1 and iron regulatory protein hepcidin in the rat kidney [23] and in human glomerular endothelial cells [44]. It has also been confirmed that treatment with ANGII could cause iron accumulation or the intracellular iron concentration, labile ferrous iron as well as ferritin induction in the rat aorta [21], kidney, heart and liver [22], and to promote NTBI uptake by bovine endothelial cells [32] and human glomerular endothelial cells [44]. The positive effect of ANGII on iron metabolism has been considered to be one of causes for ANGII-induced renal or endothelial injury, impairment of vascular function, and arterial remodeling [20]. These findings plus the results obtained from the current study indicated that there might be a regional or cellular specific regulation of ANGII on iron metabolism although the causes are unknown currently.
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The concentration of iron in the brain is region-dependent, and in the basal ganglia iron is present at a concentration equals to that found in the liver [1]. At the cell level, the oligodendrocyte is the predominant cell type that contains iron in the brain [41]. As in all cells, neurons require iron for many aspects of their physiology, including electron transport, NADPH reductase activity, myelination of axons, and as a cofactor for several enzymes involved in neurotransmitter synthesis. Hence, an imbalance in brain iron results in dysfunction in iron related metabolism [12, 43]. The regulation or management of iron at the cellular level is primarily by four proteins: TfR1, DMT1, Fpn1 and ferritin. By controlling their levels, the cell can determine the amount of iron acquired (proportional to the number of membrane TfR1 and DMT1), released (proportional to the number of membrane Fpn1) and sequestered (proportional to the cytoplasmic level of ferritin). In most types of cells, the coordinated control of TfR1, DMT1, Fpn1 and ferritin by cellular iron occurs at the post-transcriptional level and is mediated by cytoplasmic RNA binding proteins, known as the iron regulatory proteins (IRPs) [8, 17, 26], and also by the amount of a newly discovered peptide hepcidin. Currently, it is unknown whether there is a regional or cellular specific regulation of ANGII on iron transport in the brain. Also, a detailed analysis is needed to determine whether endogenous ANGII has a physiological significance or importance in brain iron metabolism. These studies are necessary for fully understanding of the mechanisms involved in bran iron homeostasis. Acknowledgments The studies in our laboratories were supported by the Competitive Earmarked Grants of The Hong Kong Research Grants Council (GRF 466713), National 973 Programs (2011CB510004), the General Grant of National Natural Science Foundation of China (NSFC) (31271132-2012, 31371092-2013), Key Project Grant of NSFC (31330035-2013) and Direct Grant of the Chinese University of Hong Kong (2011.1.084). Conflict of interest
The authors declare no conflict of interest.
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