Neuropeptides 48 (2014) 161–166
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Angiotensin II inhibits uptake of transferrin-bound iron but not non-transferrin-bound iron by cultured astrocytes Suna Huang a, Fang Du a, Lan Li a, Yong Liu b, Yuhong Liu a, Chao Zhang a, Zhong Ming Qian a,⇑ a b
Laboratory of Neuropharmacology and Department of Neurosurgery, South-west Hospital, The Third Military Medical University, Chongqing 400038, China Department of Neurology, Xin Qiao Hospital, The Third Military Medical University, Chongqing 400038, China
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Article history: Received 25 January 2014 Accepted 7 April 2014 Available online 18 April 2014 Keywords: Angiotensin II (ANGII) Brain iron metabolism Transferrin receptor 1 (TfR1) Divalent metal transporter 1 (DMT1) Ferroportin 1 (Fpn1) Transferrin-bound iron (Tf-Fe) Non-transferrin-bound iron (NTBI) Astrocytes
a b s t r a c t The existence of all components of the renin–angiotensin system (RAS) and the iron metabolism system, and the recent findings on the functions of angiotensin II (ANGII) in peripheral iron metabolism imply that ANGII might play a role in iron homeostasis by regulating expression of iron transport proteins in the brain. Here, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes. We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as the expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes. This indicated that the inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on the expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in the cells. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The primary function of the renin–angiotensin system (RAS) is to maintain fluid homeostasis and regulate blood pressure (Savaskan, 2005). Not only does it function as an endocrine system, but it also serves local paracrine and autocrine functions in tissues and organs (Mehta and Griendling, 2007). Angiotensin II (ANGII) is the primary effector molecule of this system which has emerged as a critical hormone that affects the function of virtually all organs, including heart, kidney and also brain (Dinh et al., 2001; Mehta and Griendling, 2007). In some peripheral organs and cells, a number of studies demonstrated that ANGII is able to regulate the expression of iron metabolism proteins and hence affect iron homeostasis. It has been reported that ANGII could induce a significant increase in the expression of transferrin receptor 1 (TfR1), divalent
Abbreviations: ACE, angiotensin-converting enzyme; AD, Alzheimer’s disease; ANGII, angiotensin II; DMT1 IRE, divalent metal transporter 1 without iron response element; DMT1+IRE, divalent metal transporter 1 with iron response element; Fpn1, ferroportin 1; GFAP, glial fibrillary acidic protein; NTBI, nontransferrin bound iron; PBS, phosphate buffered saline; Tf-Fe, transferrin-bound iron; TfR1, transferrin receptor 1. ⇑ Corresponding author. Tel.: +86 15618041686. E-mail address: [email protected] (Z.M. Qian). http://dx.doi.org/10.1016/j.npep.2014.04.001 0143-4179/Ó 2014 Elsevier Ltd. All rights reserved.
metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and hepcidin in rat kidney (Ishizaka et al., 2007), cause iron accumulation and ferritin induction in rat aorta (Ishizaka et al., 2005a), kidney, heart and liver (Ishizaka et al., 2005b), and promote non-transferrin-bound iron (NTBI) uptake by bovine endothelial cells (Mak et al., 2012) 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 (Tajima et al., 2010). ANGII-induced 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 (Ishizaka et al., 2002). All the required components of the RAS, including ANGII and its type 1 (AT1) and 2 (AT2) receptors (Sumners et al., 1991; Rydzewski et al., 1992; Garrido-Gil et al., 2013b), renin, angiotensinogen and angiotensin-converting enzyme (ACE) have been demonstrated to be present in the mammalian brain (Lippoldt et al., 1995; Hadjiivanova and Georgiev, 1998; Inagami et al., 1999; Gasparo et al., 2000; Mayas et al., 2005). All key proteins involved in iron metabolism and transport, such as TfR1, DMT1, Fpn1 and iron regulatory peptide hepcidin, have been found to express in the brain as well (Qian and Wang, 1998; Qian and Shen, 2001; Ke et al., 2005; Zechel et al., 2006; Wang et al., 2008). The existence
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of all components of the RAS and the iron metabolism systems, and the current understanding of ANGII functions in peripheral iron metabolism suggested strongly that ANGII might play a role to affect iron homeostasis by regulating expression of iron transport protein in the brain. A recent study by Garrido-Gil et al. (2013a) provided the first piece of convincing evidence for this hypothesis. They demonstrated that administration of angiotensin to primary mesencephalic cultures, the dopaminergic cell line MES23.5 and to young adult rats, significantly increased levels of TfR1, DMT1, and Fpn1 via type-1 receptors. In the present study, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes in vitro and found that ANGII inhibited uptake of transferrin-bound iron but not non-transferrin-bound iron in the cells.
2.4. Measurement of non-transferrin bound iron uptake The radio-labeled 55Fe(II) (NTBI) solution was prepared and the Fe(II) uptake was measured as described previously (Qian et al., 1996, 2000). After being incubated with ANGII and 55Fe(II) in 0. 27 M sucrose buffered by pH 6.5 with 4 mM Pipes, 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. 2.5. Iron release assay
2. Materials and methods 2.1. 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 transferrin receptor 1 monoclonal antibody was obtained from BD Transduction Laboratories, BD Biosciences Pharmingen, USA and antibodies against divalent metal transporter with (DMT1+IRE) or without iron response element (DMT1 IRE) and ferroportin 1 were purchased from Alpha Diagnostic International Company, San Antonio, TX, USA. The specific antibody against astrocyte glial fibrillary acidic protein (GFAP) was 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. 2.2. Primary astrocytes Primary astrocytes were prepared from 1-day-old SD rats by a procedure previously described (Qian et al., 2000; Du et al., 2011). Briefly, cerebral cortex was digested with 0.25% trypsin for 30 min at 37 °C. After trypsinization and trituration, cell suspensions were sieved through a 40-mm cell strainer and the filtrate was allowed pre-adherence for 30 min to remove any contamination from fibroblast. The plated cells were incubated in a 5% CO2 incubator (NAPCO 5400) at 37 °C. After the cultures reached confluence (12–14 days), they were subcultured 3 times at a 4-day interval and allowed pre-adherence for 30 min before being seeded in each subculture. The purity of the astrocyte cultures was assessed by staining for the astrocyte marker anti-GFAP antibodies, which was approximately 99%. 2.3. Measurement of transferrin-bound iron (Tf-Fe) uptake 55 Fe-Tf were prepared first by mixing 55FeCl3 (Perkin–Elmer) with nitrilotriacetic acid (NTA) in a 1:10 ratio and then incubating 55 Fe-NTA with apo-Tf in a 2:1 ratio for 3-h in carbonate buffer (10 mM NaHCO3, 250 mM Tris–HCl) (Qian and Morgan, 1990, 1991). After being incubated with serum-free culture medium containing 0.1% BSA at 37 °C for 1-h to remove any endogenous transferrin, astrocytes were treated with ANGII and then incubated with or without 55Fe-Tf in 0.155 M NaCl buffered by pH 7.4 with 4 mM Hepes. 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 the 55Fe taken up by the cells.
The astrocytes 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: % 55 Fe release = [(cpm in supernatant)/(cpm in supernatant + cpm in cells)] 100 (Ge et al., 2009). 2.6. Western blot analysis Astrocytes 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 Bradford assay kit (Bio-Rad). Aliquots of the cell extract containing 30 mg of protein were diluted in 2 ml sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol 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. After the transfer, the membrane was blocked with 5% blocking reagent in Tris-buffered 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 (1:1000); rabbit anti-rat DMT1+IRE, DMT1 IRE polyclonal antibodies and rabbit anti-mouse Fpn1 polyclonal antibody (1:5000). After three washes, the blots were incubated with goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody (1:5000, 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 antirat b-actin polyclonal antibody at a 1:2000 dilution (Ke et al., 2005). 2.7. Statistical analysis Statistical analyses were performed using SPSS software for Windows (version 10.0) (SPSS, Inc., Chicago, IL). 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
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of P < 0.05 was taken to be statistically significant. Correlations were analyzed by Pearson’s bivariate correlations using SPSS.
3.3. Angiotensin II suppressed expression of transferrin receptor 1 but not divalent metal transporter 1 in astrocytes
3. Results
In order to find out why ANGII can inhibit Tf-Fe and NTBI uptake by astrocytes, we investigated effects of ANGII on the expression of iron uptake proteins (TfR1, DMT1+IRE and DMT1 IRE) by pretreatment of astrocytes 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. The findings showed that treatment with 10 nM of ANGII for 12 or 24-h or 100 nM for 6, 12 or 24-h resulted in a significant decrease in the expression of TfR1 (P < 0.05 or 0.01 vs. the control) (Fig. 2A). However, no significant differences were found in the expression of DMT1+IRE (Fig. 2B) as well as DMT1 IRE (Fig. 2C) between the astrocytes treated with ANGII and the control cells, no matter lower or higher concentrations (10 or 100) and shorter or longer incubation times (6, 12 or 24-h).
3.1. Angiotensin II inhibited transferrin-bound iron but not nontransferrin bound iron uptake by cultured astrocytes We first investigated the effects of ANGII on Tf-Fe and NTBI uptake by astrocytes. The cells were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C before incubation with 55Fe(II) (1 lM) or 55Fe-Tf (10 lg/ml) for 30-min. Tf-Fe and NTBI uptake by the cells was then assayed using the radio-isotope methods. It was found that treatment with ANGII induced a remarkable decrease in TfFe uptake. The amounts of Tf-Fe (Fig. 1A) uptake in the astrocytes treated with 100 nM for 6, 12 or 24-h were 22.044 ± 3.501, 21.585 ± 2.589 and 21.734 ± 3.191 pmol/lg protein; respectively. All of these values were significantly lower than that (33.297 ± 4.586 pmol/lg protein) in the control astrocytes (P < 0.05 or 0.01). There were no significant differences between Tf-Fe uptake by astrocytes treated with ANGII 100 nM for 3-h (31.809 ± 3.389) and the control cells. However, ANGII did not induce any significant effects on NTBI uptake by astrocytes. The amounts of NTBI (Fig. 1B) uptake by the astrocytes treated with 100 nM for 3, 6, 12 or 24-h were 41.9211 ± 4.216, 42.967 ± 5.333, 43.025 ± 5.841, 42.825 ± 6.185 pmol/lg protein; respectively. There were no significant differences between all of these values and the amounts of NTBI uptake (44.877 ± 4.178 pmol/lg protein) by the control cells. 3.2. Angiotensin II inhibited iron release from the cultured astrocytes We then examined the effects of ANGII on iron release from astrocytes 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 cells was measured respectively. The percentage of 55Fe release at 6, 12 or 24-h was 41.861 ± 4.016, 54.221 ± 6.308 or 70.107 ± 2.525 in the astrocytes treated with 100 nM of ANGII, while the corresponding value in the control astrocytes at 6, 12 or 24-h was 54.861 ± 3.441, 66.078 ± 3.747 or 78.434 ± 5.791, respectively (Fig. 1C). The significant differences were found at all of these time points, indicating that ANGII could significantly inhibit iron release from the cultured astrocytes. There were no significant differences between iron release by astrocytes treated with ANGII 100 nM for 3-h (19.704 ± 5.791) and the control (20.712 ± 6.309) cells.
3.4. Angiotensin II suppressed expression of the cellular iron release protein ferroportin 1 in astrocytes Final, we investigated the effects of ANGII on the expression of iron release protein Fpn1 in order to explore the possible mechanisms of inhibition of ANGII on iron release from astrocytes. Fpn1, also known as metal transport protein 1 (MTP1), iron-regulated transporter 1 (IREG1), or Slc11a3, is the only known iron exporter in mammalian cells and plays a critical role in the maintenance of both cellular and systemic iron balance (Donovan et al., 2000). The astrocytes were treated with different concentrations of ANGII (0, 10 or 100 nM) for 0, 6, 12 or 24 h at 37 °C. Western blot analysis of Fpn1 expression was then conducted. The findings demonstrated that ANGII could induce in a significant decrease in the expression of Fpn1 (Fig. 2D). The contents of Fpn1 in astrocytes 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 its inhibitory effect on the expression of iron release protein Fpn1. 4. Discussion In the present study, we investigated effects of ANGII on iron uptake and release as well as expression of iron transport proteins in cultured astrocytes in the present study. Astrocytes were chosen for this study because the main supporting cells in the brain comprise a major class of neuroglia and perform a wide range of adap-
Fig. 1. Effects of angiotensin II (ANGII) on transferrin-bound iron (Tf-Fe) and non-transferrin bound iron (NTBI) uptake and iron release in cultured astrocytes. Astrocytes were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C before incubation with 55Fe(II) (1 lM) or 55Fe-Tf (10 lg/ml) for 30-min. To examine the effects of ANGII on iron release from astrocytes, the cells were incubated 55Fe(II) (2 lM) at 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 astrocytes were assayed as described in Section 2. All data were presented as mean ± SEM (n = 7). ⁄ P < 0.05, ⁄⁄P < 0.01 vs. the control.
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Fig. 2. Effects of angiotensin II (ANGII) on the expression of the cellular iron uptake and release proteins in astrocytes. Astrocytes were treated with 0, 10 or 100 nM of ANGII for 0, 6, 12 or 24 h 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 vs. the control.
tive functions in the mammalian nervous system (Schipper, 1996) and also because of the importance of glial cells in iron homeostasis in the brain (Chang et al., 2006). In addition, the cells play crucial roles in the maintenance of the blood–brain barrier (Wilkin et al., 1990; Schipper, 1996) as well as in proper iron handling within the central nervous system (Pelizzoni et al., 2013), and are able to accumulate iron under abnormal circumstances, e.g. in myelin-deficient rats (Connor and Menzies, 1990). We demonstrated that ANGII could significantly inhibit Tf-Fe uptake and iron release as well as expression of TfR1 and iron exporter Fpn1 in cultured astrocytes. This indicated that the significant inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on DMT1 expression as well as NTBI uptake in astrocytes, implying that no changes in NTBI uptake by astrocytes treated with ANGII is due to no effects of ANGII on DMT1 expression. Currently it is unknown how ANGII down-regulates TfR1 and Fpn1 expression and why ANGII had no effect on DMT1 expression in astrocytes. Based on the existence of ANGII type 1 (AT1) and 2 (AT2) receptors in astrocytes (Sumners et al., 1991; Rydzewski et al., 1992; Garrido-Gil et al., 2013b), however, it is possible that effect of ANGII on TfR1 expression might be mediated by AT1 and/or AT2 receptors. Further studies on this possibility are need. Our findings are different from those obtained from peripheral organs and cells. It has been reported that ANGII significantly increased expression of TfR1, DMT1 and Fpn1 and iron regulatory protein hepcidin in rat kidney (Ishizaka et al., 2007), caused iron accumulation and ferritin induction in rat aorta (Ishizaka et al., 2005a), kidney, heart and liver (Ishizaka et al., 2005b) and facilitated NTBI uptake by bovine endothelial cells (Mak et al.,
2012), expression of iron uptake and release proteins, and the intracellular iron concentration as well as labile ferrous iron in human glomerular endothelial cells (Tajima et al., 2010). The difference between these studies and ours might suggest the existence of a regional or cellular specific regulation of ANGII on iron metabolism. This possibility needs to be further investigated. In a recent study, Garrido-Gil et al. (2013a) demonstrated that ANGII could induce a significant increase in TfR1, DMT1, Fpn1, ferritin and labile iron in primary mesencephalic cultures (>85% neuron) and the dopaminergic cell line MES23.5. They also found that ANGII did not induce significant changes in levels of ferritin or labile iron in primary neuron-glia cultures. In the present study, however, ANGII was found to significantly decrease, rather than increase, expression of TfR1 and Fpn1 and have no effect on expression of DMT1 in cultured astrocytes. The causes led to the difference in the response to ANGII in different types of brain cells are unknown currently. However, it has been well documented that the distribution of iron in different types of cells in the brain is different (Rebeck et al., 1995). This fact plus the above findings suggested that there might be a cell-specific effect of ANGII in the brain. In the last decades, a large number of experimental studies had demonstrated that ANGII is a key factor in the inflammatory process (Suzuki et al., 2003; Agarwal et al., 2013). It has also been reported that inflammatory cytokines increase NTBI uptake by astrocytes as well as DMT1 expression. Urrutia et al. (2013) found that inflammatory stimuli (tumor necrosis factor alpha, TNF-a; interleukin 6, IL-6 and lipopolysaccharide, LPS) increased expression of DMT1 in astrocytes in addition to neurons and microglia, and also induced the expression of hepcidin in astrocytes and microglia, but not in neurons. The net result of these changes
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was the increased iron accumulation in neurons and microglia but not in astrocytes. Pelizzoni et al. (2013) investigated the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes and confirmed that the main source of iron is NTBI. They also showed that expression of all DMT1 isoforms is increased in astrocytes exposed to TNFa and IL1b by RT-qPCR analysis. In the present study, we did not measure the effect of ANGII on hepcidin expression. However, the increased expression of hepcidin induced by inflammatory stimuli in astrocytes (Urrutia et al., 2013) might be able to partly explain why Fpn1 level decreased in ANGII-treated cells found in our study. In addition, further studies are needed to investigate why DMT1 expression is increased in astrocytes exposed to inflammatory cytokines IL1b and TNFa (Pelizzoni et al., 2013) but no change in astrocytes exposed to ANGII. Iron is an essential element that is required for fundamental cell functions in all living cells including astrocytes (Qian et al., 1997; Qian and Wang, 1998). 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 considered as a primary cause of neurodegenerative disorders (Ke and Qian, 2003, 2007). Therefore, the fully understanding of homeostatic mechanisms involved in brain iron metabolism is fundamental and critical not only for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain but also for developing pharmacological interventions that can disrupt the chain of pathological events occurring in neurodegenerative diseases caused by iron accumulation. At the cell level, the oligodendrocyte is the predominant cell type containing iron in the brain (Rebeck et al., 1995). Therefore, further studies will be absolutely needed to elucidate the effects of ANGII on cell iron transport proteins and their relevant mechanisms involved in oligodendrocyte. Also, it is critical to study whether ANGII has an effect on the expression of iron regulate proteins (IRPs) in the brain. These studies will help us to answer the question of whether ANGII is a physiological regulator in brain iron metabolism. 5. Conclusion We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes in vitro. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in astrocytes in vitro. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis. Author contributions Z.M.Q. conceived, organized and supervised the study; S.N.H., F.D., L.L., Y.L., Y.H.L. and C.Z. performed the experiments; S.N.H. and F.D. contributed to the analysis and interpretation of data. Z.M.Q. prepared and wrote the manuscript. Acknowledgments The studies in our laboratories were supported by National 973 Programs (2011CB510004), the General Grant of National Natural Science Foundation of China (NSFC) (31271132-2012) and Key Project Grant of NSFC (31330035-2013).
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Angiotensin II inhibits uptake of transferrin-bound iron but not non-transferrin-bound iron by cultured astrocytes Suna Huang a, Fang Du a, Lan Li a, Yong Liu b, Yuhong Liu a, Chao Zhang a, Zhong Ming Qian a,⇑ a b
Laboratory of Neuropharmacology and Department of Neurosurgery, South-west Hospital, The Third Military Medical University, Chongqing 400038, China Department of Neurology, Xin Qiao Hospital, The Third Military Medical University, Chongqing 400038, China
a r t i c l e
i n f o
Article history: Received 25 January 2014 Accepted 7 April 2014 Available online 18 April 2014 Keywords: Angiotensin II (ANGII) Brain iron metabolism Transferrin receptor 1 (TfR1) Divalent metal transporter 1 (DMT1) Ferroportin 1 (Fpn1) Transferrin-bound iron (Tf-Fe) Non-transferrin-bound iron (NTBI) Astrocytes
a b s t r a c t The existence of all components of the renin–angiotensin system (RAS) and the iron metabolism system, and the recent findings on the functions of angiotensin II (ANGII) in peripheral iron metabolism imply that ANGII might play a role in iron homeostasis by regulating expression of iron transport proteins in the brain. Here, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes. We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as the expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes. This indicated that the inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on the expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in the cells. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The primary function of the renin–angiotensin system (RAS) is to maintain fluid homeostasis and regulate blood pressure (Savaskan, 2005). Not only does it function as an endocrine system, but it also serves local paracrine and autocrine functions in tissues and organs (Mehta and Griendling, 2007). Angiotensin II (ANGII) is the primary effector molecule of this system which has emerged as a critical hormone that affects the function of virtually all organs, including heart, kidney and also brain (Dinh et al., 2001; Mehta and Griendling, 2007). In some peripheral organs and cells, a number of studies demonstrated that ANGII is able to regulate the expression of iron metabolism proteins and hence affect iron homeostasis. It has been reported that ANGII could induce a significant increase in the expression of transferrin receptor 1 (TfR1), divalent
Abbreviations: ACE, angiotensin-converting enzyme; AD, Alzheimer’s disease; ANGII, angiotensin II; DMT1 IRE, divalent metal transporter 1 without iron response element; DMT1+IRE, divalent metal transporter 1 with iron response element; Fpn1, ferroportin 1; GFAP, glial fibrillary acidic protein; NTBI, nontransferrin bound iron; PBS, phosphate buffered saline; Tf-Fe, transferrin-bound iron; TfR1, transferrin receptor 1. ⇑ Corresponding author. Tel.: +86 15618041686. E-mail address: [email protected] (Z.M. Qian). http://dx.doi.org/10.1016/j.npep.2014.04.001 0143-4179/Ó 2014 Elsevier Ltd. All rights reserved.
metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and hepcidin in rat kidney (Ishizaka et al., 2007), cause iron accumulation and ferritin induction in rat aorta (Ishizaka et al., 2005a), kidney, heart and liver (Ishizaka et al., 2005b), and promote non-transferrin-bound iron (NTBI) uptake by bovine endothelial cells (Mak et al., 2012) 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 (Tajima et al., 2010). ANGII-induced 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 (Ishizaka et al., 2002). All the required components of the RAS, including ANGII and its type 1 (AT1) and 2 (AT2) receptors (Sumners et al., 1991; Rydzewski et al., 1992; Garrido-Gil et al., 2013b), renin, angiotensinogen and angiotensin-converting enzyme (ACE) have been demonstrated to be present in the mammalian brain (Lippoldt et al., 1995; Hadjiivanova and Georgiev, 1998; Inagami et al., 1999; Gasparo et al., 2000; Mayas et al., 2005). All key proteins involved in iron metabolism and transport, such as TfR1, DMT1, Fpn1 and iron regulatory peptide hepcidin, have been found to express in the brain as well (Qian and Wang, 1998; Qian and Shen, 2001; Ke et al., 2005; Zechel et al., 2006; Wang et al., 2008). The existence
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of all components of the RAS and the iron metabolism systems, and the current understanding of ANGII functions in peripheral iron metabolism suggested strongly that ANGII might play a role to affect iron homeostasis by regulating expression of iron transport protein in the brain. A recent study by Garrido-Gil et al. (2013a) provided the first piece of convincing evidence for this hypothesis. They demonstrated that administration of angiotensin to primary mesencephalic cultures, the dopaminergic cell line MES23.5 and to young adult rats, significantly increased levels of TfR1, DMT1, and Fpn1 via type-1 receptors. In the present study, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes in vitro and found that ANGII inhibited uptake of transferrin-bound iron but not non-transferrin-bound iron in the cells.
2.4. Measurement of non-transferrin bound iron uptake The radio-labeled 55Fe(II) (NTBI) solution was prepared and the Fe(II) uptake was measured as described previously (Qian et al., 1996, 2000). After being incubated with ANGII and 55Fe(II) in 0. 27 M sucrose buffered by pH 6.5 with 4 mM Pipes, 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. 2.5. Iron release assay
2. Materials and methods 2.1. 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 transferrin receptor 1 monoclonal antibody was obtained from BD Transduction Laboratories, BD Biosciences Pharmingen, USA and antibodies against divalent metal transporter with (DMT1+IRE) or without iron response element (DMT1 IRE) and ferroportin 1 were purchased from Alpha Diagnostic International Company, San Antonio, TX, USA. The specific antibody against astrocyte glial fibrillary acidic protein (GFAP) was 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. 2.2. Primary astrocytes Primary astrocytes were prepared from 1-day-old SD rats by a procedure previously described (Qian et al., 2000; Du et al., 2011). Briefly, cerebral cortex was digested with 0.25% trypsin for 30 min at 37 °C. After trypsinization and trituration, cell suspensions were sieved through a 40-mm cell strainer and the filtrate was allowed pre-adherence for 30 min to remove any contamination from fibroblast. The plated cells were incubated in a 5% CO2 incubator (NAPCO 5400) at 37 °C. After the cultures reached confluence (12–14 days), they were subcultured 3 times at a 4-day interval and allowed pre-adherence for 30 min before being seeded in each subculture. The purity of the astrocyte cultures was assessed by staining for the astrocyte marker anti-GFAP antibodies, which was approximately 99%. 2.3. Measurement of transferrin-bound iron (Tf-Fe) uptake 55 Fe-Tf were prepared first by mixing 55FeCl3 (Perkin–Elmer) with nitrilotriacetic acid (NTA) in a 1:10 ratio and then incubating 55 Fe-NTA with apo-Tf in a 2:1 ratio for 3-h in carbonate buffer (10 mM NaHCO3, 250 mM Tris–HCl) (Qian and Morgan, 1990, 1991). After being incubated with serum-free culture medium containing 0.1% BSA at 37 °C for 1-h to remove any endogenous transferrin, astrocytes were treated with ANGII and then incubated with or without 55Fe-Tf in 0.155 M NaCl buffered by pH 7.4 with 4 mM Hepes. 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 the 55Fe taken up by the cells.
The astrocytes 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: % 55 Fe release = [(cpm in supernatant)/(cpm in supernatant + cpm in cells)] 100 (Ge et al., 2009). 2.6. Western blot analysis Astrocytes 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 Bradford assay kit (Bio-Rad). Aliquots of the cell extract containing 30 mg of protein were diluted in 2 ml sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol 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. After the transfer, the membrane was blocked with 5% blocking reagent in Tris-buffered 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 (1:1000); rabbit anti-rat DMT1+IRE, DMT1 IRE polyclonal antibodies and rabbit anti-mouse Fpn1 polyclonal antibody (1:5000). After three washes, the blots were incubated with goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody (1:5000, 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 antirat b-actin polyclonal antibody at a 1:2000 dilution (Ke et al., 2005). 2.7. Statistical analysis Statistical analyses were performed using SPSS software for Windows (version 10.0) (SPSS, Inc., Chicago, IL). 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
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of P < 0.05 was taken to be statistically significant. Correlations were analyzed by Pearson’s bivariate correlations using SPSS.
3.3. Angiotensin II suppressed expression of transferrin receptor 1 but not divalent metal transporter 1 in astrocytes
3. Results
In order to find out why ANGII can inhibit Tf-Fe and NTBI uptake by astrocytes, we investigated effects of ANGII on the expression of iron uptake proteins (TfR1, DMT1+IRE and DMT1 IRE) by pretreatment of astrocytes 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. The findings showed that treatment with 10 nM of ANGII for 12 or 24-h or 100 nM for 6, 12 or 24-h resulted in a significant decrease in the expression of TfR1 (P < 0.05 or 0.01 vs. the control) (Fig. 2A). However, no significant differences were found in the expression of DMT1+IRE (Fig. 2B) as well as DMT1 IRE (Fig. 2C) between the astrocytes treated with ANGII and the control cells, no matter lower or higher concentrations (10 or 100) and shorter or longer incubation times (6, 12 or 24-h).
3.1. Angiotensin II inhibited transferrin-bound iron but not nontransferrin bound iron uptake by cultured astrocytes We first investigated the effects of ANGII on Tf-Fe and NTBI uptake by astrocytes. The cells were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C before incubation with 55Fe(II) (1 lM) or 55Fe-Tf (10 lg/ml) for 30-min. Tf-Fe and NTBI uptake by the cells was then assayed using the radio-isotope methods. It was found that treatment with ANGII induced a remarkable decrease in TfFe uptake. The amounts of Tf-Fe (Fig. 1A) uptake in the astrocytes treated with 100 nM for 6, 12 or 24-h were 22.044 ± 3.501, 21.585 ± 2.589 and 21.734 ± 3.191 pmol/lg protein; respectively. All of these values were significantly lower than that (33.297 ± 4.586 pmol/lg protein) in the control astrocytes (P < 0.05 or 0.01). There were no significant differences between Tf-Fe uptake by astrocytes treated with ANGII 100 nM for 3-h (31.809 ± 3.389) and the control cells. However, ANGII did not induce any significant effects on NTBI uptake by astrocytes. The amounts of NTBI (Fig. 1B) uptake by the astrocytes treated with 100 nM for 3, 6, 12 or 24-h were 41.9211 ± 4.216, 42.967 ± 5.333, 43.025 ± 5.841, 42.825 ± 6.185 pmol/lg protein; respectively. There were no significant differences between all of these values and the amounts of NTBI uptake (44.877 ± 4.178 pmol/lg protein) by the control cells. 3.2. Angiotensin II inhibited iron release from the cultured astrocytes We then examined the effects of ANGII on iron release from astrocytes 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 cells was measured respectively. The percentage of 55Fe release at 6, 12 or 24-h was 41.861 ± 4.016, 54.221 ± 6.308 or 70.107 ± 2.525 in the astrocytes treated with 100 nM of ANGII, while the corresponding value in the control astrocytes at 6, 12 or 24-h was 54.861 ± 3.441, 66.078 ± 3.747 or 78.434 ± 5.791, respectively (Fig. 1C). The significant differences were found at all of these time points, indicating that ANGII could significantly inhibit iron release from the cultured astrocytes. There were no significant differences between iron release by astrocytes treated with ANGII 100 nM for 3-h (19.704 ± 5.791) and the control (20.712 ± 6.309) cells.
3.4. Angiotensin II suppressed expression of the cellular iron release protein ferroportin 1 in astrocytes Final, we investigated the effects of ANGII on the expression of iron release protein Fpn1 in order to explore the possible mechanisms of inhibition of ANGII on iron release from astrocytes. Fpn1, also known as metal transport protein 1 (MTP1), iron-regulated transporter 1 (IREG1), or Slc11a3, is the only known iron exporter in mammalian cells and plays a critical role in the maintenance of both cellular and systemic iron balance (Donovan et al., 2000). The astrocytes were treated with different concentrations of ANGII (0, 10 or 100 nM) for 0, 6, 12 or 24 h at 37 °C. Western blot analysis of Fpn1 expression was then conducted. The findings demonstrated that ANGII could induce in a significant decrease in the expression of Fpn1 (Fig. 2D). The contents of Fpn1 in astrocytes 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 its inhibitory effect on the expression of iron release protein Fpn1. 4. Discussion In the present study, we investigated effects of ANGII on iron uptake and release as well as expression of iron transport proteins in cultured astrocytes in the present study. Astrocytes were chosen for this study because the main supporting cells in the brain comprise a major class of neuroglia and perform a wide range of adap-
Fig. 1. Effects of angiotensin II (ANGII) on transferrin-bound iron (Tf-Fe) and non-transferrin bound iron (NTBI) uptake and iron release in cultured astrocytes. Astrocytes were treated with 100 nM of ANGII for 0, 3, 6, 12 or 24-h at 37 °C before incubation with 55Fe(II) (1 lM) or 55Fe-Tf (10 lg/ml) for 30-min. To examine the effects of ANGII on iron release from astrocytes, the cells were incubated 55Fe(II) (2 lM) at 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 astrocytes were assayed as described in Section 2. All data were presented as mean ± SEM (n = 7). ⁄ P < 0.05, ⁄⁄P < 0.01 vs. the control.
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Fig. 2. Effects of angiotensin II (ANGII) on the expression of the cellular iron uptake and release proteins in astrocytes. Astrocytes were treated with 0, 10 or 100 nM of ANGII for 0, 6, 12 or 24 h 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 vs. the control.
tive functions in the mammalian nervous system (Schipper, 1996) and also because of the importance of glial cells in iron homeostasis in the brain (Chang et al., 2006). In addition, the cells play crucial roles in the maintenance of the blood–brain barrier (Wilkin et al., 1990; Schipper, 1996) as well as in proper iron handling within the central nervous system (Pelizzoni et al., 2013), and are able to accumulate iron under abnormal circumstances, e.g. in myelin-deficient rats (Connor and Menzies, 1990). We demonstrated that ANGII could significantly inhibit Tf-Fe uptake and iron release as well as expression of TfR1 and iron exporter Fpn1 in cultured astrocytes. This indicated that the significant inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on DMT1 expression as well as NTBI uptake in astrocytes, implying that no changes in NTBI uptake by astrocytes treated with ANGII is due to no effects of ANGII on DMT1 expression. Currently it is unknown how ANGII down-regulates TfR1 and Fpn1 expression and why ANGII had no effect on DMT1 expression in astrocytes. Based on the existence of ANGII type 1 (AT1) and 2 (AT2) receptors in astrocytes (Sumners et al., 1991; Rydzewski et al., 1992; Garrido-Gil et al., 2013b), however, it is possible that effect of ANGII on TfR1 expression might be mediated by AT1 and/or AT2 receptors. Further studies on this possibility are need. Our findings are different from those obtained from peripheral organs and cells. It has been reported that ANGII significantly increased expression of TfR1, DMT1 and Fpn1 and iron regulatory protein hepcidin in rat kidney (Ishizaka et al., 2007), caused iron accumulation and ferritin induction in rat aorta (Ishizaka et al., 2005a), kidney, heart and liver (Ishizaka et al., 2005b) and facilitated NTBI uptake by bovine endothelial cells (Mak et al.,
2012), expression of iron uptake and release proteins, and the intracellular iron concentration as well as labile ferrous iron in human glomerular endothelial cells (Tajima et al., 2010). The difference between these studies and ours might suggest the existence of a regional or cellular specific regulation of ANGII on iron metabolism. This possibility needs to be further investigated. In a recent study, Garrido-Gil et al. (2013a) demonstrated that ANGII could induce a significant increase in TfR1, DMT1, Fpn1, ferritin and labile iron in primary mesencephalic cultures (>85% neuron) and the dopaminergic cell line MES23.5. They also found that ANGII did not induce significant changes in levels of ferritin or labile iron in primary neuron-glia cultures. In the present study, however, ANGII was found to significantly decrease, rather than increase, expression of TfR1 and Fpn1 and have no effect on expression of DMT1 in cultured astrocytes. The causes led to the difference in the response to ANGII in different types of brain cells are unknown currently. However, it has been well documented that the distribution of iron in different types of cells in the brain is different (Rebeck et al., 1995). This fact plus the above findings suggested that there might be a cell-specific effect of ANGII in the brain. In the last decades, a large number of experimental studies had demonstrated that ANGII is a key factor in the inflammatory process (Suzuki et al., 2003; Agarwal et al., 2013). It has also been reported that inflammatory cytokines increase NTBI uptake by astrocytes as well as DMT1 expression. Urrutia et al. (2013) found that inflammatory stimuli (tumor necrosis factor alpha, TNF-a; interleukin 6, IL-6 and lipopolysaccharide, LPS) increased expression of DMT1 in astrocytes in addition to neurons and microglia, and also induced the expression of hepcidin in astrocytes and microglia, but not in neurons. The net result of these changes
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was the increased iron accumulation in neurons and microglia but not in astrocytes. Pelizzoni et al. (2013) investigated the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes and confirmed that the main source of iron is NTBI. They also showed that expression of all DMT1 isoforms is increased in astrocytes exposed to TNFa and IL1b by RT-qPCR analysis. In the present study, we did not measure the effect of ANGII on hepcidin expression. However, the increased expression of hepcidin induced by inflammatory stimuli in astrocytes (Urrutia et al., 2013) might be able to partly explain why Fpn1 level decreased in ANGII-treated cells found in our study. In addition, further studies are needed to investigate why DMT1 expression is increased in astrocytes exposed to inflammatory cytokines IL1b and TNFa (Pelizzoni et al., 2013) but no change in astrocytes exposed to ANGII. Iron is an essential element that is required for fundamental cell functions in all living cells including astrocytes (Qian et al., 1997; Qian and Wang, 1998). 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 considered as a primary cause of neurodegenerative disorders (Ke and Qian, 2003, 2007). Therefore, the fully understanding of homeostatic mechanisms involved in brain iron metabolism is fundamental and critical not only for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain but also for developing pharmacological interventions that can disrupt the chain of pathological events occurring in neurodegenerative diseases caused by iron accumulation. At the cell level, the oligodendrocyte is the predominant cell type containing iron in the brain (Rebeck et al., 1995). Therefore, further studies will be absolutely needed to elucidate the effects of ANGII on cell iron transport proteins and their relevant mechanisms involved in oligodendrocyte. Also, it is critical to study whether ANGII has an effect on the expression of iron regulate proteins (IRPs) in the brain. These studies will help us to answer the question of whether ANGII is a physiological regulator in brain iron metabolism. 5. Conclusion We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes in vitro. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in astrocytes in vitro. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis. Author contributions Z.M.Q. conceived, organized and supervised the study; S.N.H., F.D., L.L., Y.L., Y.H.L. and C.Z. performed the experiments; S.N.H. and F.D. contributed to the analysis and interpretation of data. Z.M.Q. prepared and wrote the manuscript. Acknowledgments The studies in our laboratories were supported by National 973 Programs (2011CB510004), the General Grant of National Natural Science Foundation of China (NSFC) (31271132-2012) and Key Project Grant of NSFC (31330035-2013).
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