Neuroglobin and cytoglobin expression in mice Evidence for a correlation with reactive oxygen species scavenging Elke Fordel, Liesbet Thijs, Luc Moens and Sylvia Dewilde Department of Biomedical Sciences, University of Antwerp, Belgium Both hypoxia (lack of O 2 relative to metabolic needs) and reoxygenation (reintroduction of O 2 to hypoxic tissue) are important in human pathophysiology. Prominent examples of tissue hypoxia that predispose to injury during reoxygenation include myocardial isc- hemia, stroke and transplantation of organs. Under- standing the role of reactive oxygen species (ROS) in reoxygenation injury has the potential to lead to ther- apies that could improve public health [1]. Although ROS at physiologic concentrations may be required for normal cell function, excessive production of ROS is detrimental to cells, because ROS cause oxidative damage to lipids, proteins, and DNA. All cells possess a complex antioxidant system to detoxify ROS. Under normal conditions, antioxidant enzymes act in concert to remove various ROS produced by free radical reac- tions (Cu ⁄ Zn-superoxide dismutase and Mn-superoxide dismutase, catalase, and glutathione peroxidase) [1]. Neuroglobin (Ngb) and cytoglobin (Cygb) are two new members of the globin family. Ngb is mainly expressed in the cytoplasm of neuronal (brain and retina) and some endocrine tissues, whereas Cygb is detected in the nucleus and in the cytoplasm of many types of tissue [2,3]. In response to hypoxia, Ngb is up- regulated in vitro [4,5], and protects neurons against hypoxic damage. However, there are contradictory data as to whether Ngb is upregulated under hypoxic conditions in brain [4,6,7]. Several factors, namely spe- cies, brain region, severity and duration of hypoxia, and the pattern of hypoxic exposures, could have accounted for such disparate findings [8]. Cygb is up- regulated in vitro [4] and in vivo under hypoxia [4,9]. Disparities between Ngb and Cygb responses to hyp- oxia suggest that these two globins may play different roles. Recently, a possible role in ROS detoxification was suggested for Ngb and Cygb in brain and retina. Keywords cytoglobin; eyes; hypoxia; neuroglobin; reactive oxygen species Correspondence S. Dewilde, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium Fax: +32 3 8202248 Tel: +32 3 8202392 E-mail: sylvia.dewilde@ua.ac.be (Received 29 September 2006, revised 7 December 2006, accepted 4 January 2007) doi:10.1111/j.1742-4658.2007.05679.x Although essentially unknown, several functions are hypothesized for neu- roglobin and cytoglobin, two new members of the globin family. In this article, we try to shed more light on their possible roles in hypoxia and detoxification of reactive oxygen species in vivo. The relative transcriptional changes of neuroglobin and cytoglobin in a situation of chronic hypoxia in mice were examined using real-time quantitative PCR. The kinetics of the hypoxic expression of neuroglobin (brain, eyes) and cytoglobin (brain, eyes, liver, heart, skeletal muscle) is organ-specific. Moreover, reactive oxygen species production is higher in liver than in the other tissues. In eyes, the negative correlation, after reoxygenation, between neuroglobin protein level and H 2 O 2 concentration is a first proof of a reactive oxygen species- scavenging function for neuroglobin. In addition, apoptotic cell death after hypoxia is for the first time demonstrated in heart and liver. Abbreviations Cygb, cytoglobin; HIF, hypoxia-inducible factor; Ngb, neuroglobin; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor. 1312 FEBS Journal 274 (2007) 1312–1317 ª 2007 The Authors Journal compilation ª 2007 FEBS Fago et al. [10] described a mathematical model of ret- inal O 2 supply that argues that Ngb plays a role in scavenging ROS and reactive nitrogen species that are generated following hypoxia. Ostojic et al. [11] tried to find an explanation for why the retina is capable of tolerating an almost 10 times longer period of ischemia than brain. They described how the presence of hypo- xia in the retina is consistent with the finding of increased hypoxia-inducible factor (HIF)-1a and up- regulation of Cygb mRNA, and hypothesized that Cygb (and Ngb) may have a vital role in retinal oxy- gen homeostasis, enabling the retina to sustain longer periods of ischemia. Our previous study [4] showed that Cygb is signifi- cantly upregulated upon 48 h of hypoxia, and that this increase is dependent on the tissue and the severity of hypoxia. The mechanism of induction of Cygb is regu- lated by HIF-1. The aim of this study was to illustrate the time dependency and the tissue specificity of Cygb and Ngb mRNA upregulation upon hypoxia and after reoxygenation. In addition, we tried to shed light on the ROS-scavenging function of Ngb and on the mech- anism of cell death after hypoxic exposure. Results and Discussion As can be seen in Fig. 1, the mRNA upregulation is dependent on the hypoxia exposure time and is tissue- specific. Vascular endothelial growth factor (VEGF) [12] and Mb [13] were used as positive controls, as they are both known to be regulated by hypoxia. As expected, the HIF-1a upregulation was negligible in all tissues, which confirms that HIF-1a is not transcrip- tionally regulated, but is rather regulated at the post- mRNA level [14]. However, in liver, statistically signifi- cant upregulation after 48 h of hypoxia ± 8 h of reoxygenation can be seen. The liver is unique because of its physiologic O 2 gradient [15]. Under normal con- ditions, periportal (pO 2 high) and perivenous (pO 2 low) cells need the same levels of HIF proteins, but under abnormal conditions leading to hypoxia the perivenous cells need more HIF proteins within short periods of time. Therefore, they contain higher levels of HIF-a mRNA, so the HIF-a proteins can be eleva- ted rapidly by de novo protein synthesis using pre-exist- ing mRNA [15]. This could possibly explain the higher level of HIF-1a mRNA after long-term hypoxia and short-term reoxygenation, in comparison with the other tissues (Fig. 1). Cygb upregulation gradually increases as a function of time, and reaches maximal levels after 48 h of hyp- oxia in brain, heart, and liver. For muscle, the highest upregulation is also obtained after 48 h, but a drop can be seen around 12 h, although it is not statistically significant. For eyes, however, Cygb is maximally expressed around 24 h. After reoxygenation, the upregulation gradually decreases for all tissues; the slowest decreases are in liver and heart. Ngb expres- sion follows the same trends. In heart, Cygb mRNA upregulation only starts after 48 h of hypoxia. This could be due to the presence of mechanisms for self-protection under the pernicious conditions of hypoxia [16]. These mechanisms are directed towards a restoration of the normal ratio of energy demand versus energy supply [16]. In eyes, the upregulation of Ngb is higher than that of VEGF, and starts earlier than in the other tissues. The retina is extremely prone to oxidative damage, owing to its relatively high O 2 consumption and its constant exposure to light. Oxidative stress is thought to be involved in the pathogenesis of many ocular dis- eases [17]. Schmidt et al. [18] showed that Ngb is highly expressed in the murine retina, and that the intraretinal distribution of Ngb correlates with the relative oxygen consumption. Although still a matter of debate, the estimated local concentration of Ngb in the mouse retina is > 100 lm, and is thus within the range of Mb in the muscle, indicating the important role of Ngb in retinal function [18]. However, in con- trast to Mb, the O 2 affinity of Ngb is markedly sensi- tive to changes in pH and temperature [10]. Under physiologic conditions, the O 2 affinity is much lower (P 50 7.5 versus 1), whereby only a small fraction of Ngb would be saturated with O 2 under normal condi- tions. According to a mathematical model, Ngb would therefore improve O 2 supply only at concentrations exceeding 300 lm [10]. A more likely role appears to be cellular detoxification of free radicals and related oxidizing species. To unravel the possible ROS-scavenging function of Ngb and Cygb, ROS levels, expressed as pmol H 2 O 2 (Fig. 2), were followed in first order as a function of time for the different tissues. The obtained values for eyes, brain, muscle and heart were all in the same range (1–11 pmol), whereas the values for liver were all higher (18–35 pmol). This is to be expected, as the liver is more tolerant to hypoxia [15] and thus more tolerant to higher H 2 O 2 levels. For brain and eyes, there was a gradual increase in the amount of H 2 O 2 after hypoxia and reoxygenation. In heart, muscle and liver, the H 2 O 2 level dropped after 24 h of reoxygena- tion. The correlation between ROS detection and pro- tein level was studied in second order. As can be seen in Fig. 3, in eyes, during hypoxic incubation, H 2 O 2 production remained at levels comparable with norm- oxia. This finding was expected, as it is reoxygenation, E. Fordel et al. Neuroglobin as ROS scavenger? FEBS Journal 274 (2007) 1312–1317 ª 2007 The Authors Journal compilation ª 2007 FEBS 1313 rather than hypoxia, that induces ROS production (reperfusion injury). After 48 h of hypoxia, Ngb pro- tein expression, relative to normoxia, was upregulated almost two-fold. In comparison to 48 h of hypoxia, the Ngb protein upregulation decreased after reoxygen- ation, whereas the H 2 O 2 level increased. This negative correlation can be interpreted as a scavenger function for Ngb. For Ngb in brain, this negative correlation was less clear. This result is in agreement with previous in vitro experiments in our laboratory [19], which clearly indicated that Ngb protects human SH-SY5Y neuroblastoma cells from H 2 O 2 -induced cell death. Herold et al. [20] also studied the in vitro reaction of Ngb with H 2 O 2 ,NO Æ and peroxynitrite, and showed that Ngb is an efficient scavenger of ROS. Upon hyp- oxia ⁄ reperfusion, NO Æ production rises dramatically up to low micromolar levels [21], and the superoxide level is increased in neurons [22]. Although most NO Æ is rap- idly scavenged by Hb, acute endogenously produced NO Æ can be scavenged by Ngb, depending on the ligation and oxidation state of the iron center of the heme. A major fate of the excess NO Æ appears to AB C E D Fig. 1. Absolute mRNA upregulation in mice under hypoxic conditions and after reoxygenation. Real-time quantitative PCR results of the absolute mRNA upregulation of VEGF, Mb, HIF-1a, Ngb and Cygb in eyes (A), brain (B), skeletal muscle (C), heart (D), and liver (E). 48R8, 48R24: 48 h of hypoxia, followed by 8 h or 24 h of reoxygenation. Data are expressed as mean ± SEM of two independent experiments, each with three mice. *Statistically significant difference from the reference normoxic condition (P ¼ 0.05). Neuroglobin as ROS scavenger? E. Fordel et al. 1314 FEBS Journal 274 (2007) 1312–1317 ª 2007 The Authors Journal compilation ª 2007 FEBS be the reaction with superoxide to form peroxynitrite. In the Fe 2+ –NO form, Ngb reacts more rapidly with ONOO – than does Hb. In contrast to Hb and Mb, the reaction of met(Fe 3+ ) Ngb with ONOO – or H 2 O 2 does not appear to generate the cytotoxic ferryl (Fe 4+ ) spe- cies, which results in the formation of secondary pro- tein radicals. This feature of Ngb may be related to the hexacoordinated heme [21]. The ROS-scavenging reac- tion of Ngb may thus contribute to neuronal survival following hypoxic ⁄ ischemic episodes, provided that the heme is in the Fe 2+ –NO form. This appears plausible at least in the retina, where relatively high levels of Ngb are localized [18]. As well as the heme, other components may also contribute to ROS scavenging, such as reactive thiols, which are readily accessible to oxidizing agents [21]. The exact mechanism by which Ngb protects against oxidative stress remains to be elu- cidated. Investigation of mechanisms of cell death during cell injury in vivo caused by ischemia and reperfusion is of clinical importance, but technically difficult. In heart and liver, apoptotic cell death is demonstrated for the first time by incubation with the cleaved caspase-3 antibody (Fig. 4). Caspase-3 is one of the key executers of apoptosis, as it is responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly(ADP-ribose) polymerase. The highest sig- nal is seen after 48 h of hypoxia, and it gradually declines during reoxygenation. Communal et al. [23] and Liu et al. [24] described the important role of apoptosis in heart and liver diseases, respectively. In skeletal muscle, brain and eyes, apoptotic cell death could not be demonstrated. Hypoxia ⁄ ischemia may simultaneously activate multiple mechanisms of cell death to varying degrees, resulting in a spectrum of morphologic changes ranging from apoptosis to necro- sis [25], and may thus not be clearly detectable on western blot in these organs. Experimental procedures Induction of hypoxia Swiss-CD1 mice were kept in a chamber maintained at 7% O 2 (premixed gases: 7% O 2 , 93% N 2 ; Messer, Machelen, Belgium). Mice were provided with food and water ad libitum, and allowed to adjust to the hypoxic environ- ment by gradually decreasing O 2 from 21% (normoxia) to 7% (hypoxia) over an adaptation time of 1 h. After 2 h, 4 h, 6 h, 12 h, 24 h and 48 h of hypoxic exposure, the ani- 0 10 20 30 40 50 eyes brain * * * * muscle heart liver H 2 O 2 level (pmol) N H48 48R8 48R24 Fig. 2. H 2 O 2 level measured in mice under hypoxic conditions and after reoxygenation. Evolution of ROS detection, expressed as H 2 O 2 level, in different tissues when mice are exposed to normoxia (N) or hypoxia (H) for 48 h, and after reoxygenation. 48R8, 48R24: 48 h of hypoxia, followed by 8 h or 24 h of reoxygenation. Data are expressed as mean ± SEM of three independent experiments. *Statistically significant difference from the normoxic condition (P ¼ 0.05). Fig. 3. Relative Ngb protein expression and H 2 O 2 level in mice under hypoxic conditions and after reoxygenation. Comparison between Ngb protein expression (normoxia ¼ 100%, normalization with b-actin) and H 2 O 2 level to investigate the ROS-scavenging function of Ngb in eyes. H: hypoxia. 48R8, 48R24: 48 h of hypoxia, followed by 8 h or 24 h of reoxygenation. Fig. 4. Western blot detection in mice under hypoxic conditions and after reoxygenation. Cleaved caspase-3 protein expression in heart and liver under hypoxic (H) conditions (48 h) and after reoxy- genation (48R8, 48R24: 48 h of hypoxia, followed by 8 h or 24 h of reoxygenation). Positive control: U937 monocytes treated with 50 l M etoposide for 5 h. E. Fordel et al. Neuroglobin as ROS scavenger? FEBS Journal 274 (2007) 1312–1317 ª 2007 The Authors Journal compilation ª 2007 FEBS 1315 mals were killed, and the dissected organs (brain, liver, heart, skeletal muscle, eyes) were frozen in liquid nitrogen and stored at ) 80 °C until further processing. The influence of reoxygenation (8 h and 24 h) was tested after 48 h of hypoxia. All procedures were approved by the local ethics committee of the University of Antwerp, and conformed to European Community regulations. RNA extraction and real-time quantitative PCR For RNA extraction, the TriZol method (Invitrogen, Carls- bad, CA, USA) was used, followed by LiCl precipitation (Ambion, Austin, TX, USA). cDNA was synthesized using random primers (Promega, Madison, WI, USA) and Super- script II RT enzyme according to the manufacturer’s instructions (Invitrogen). Real-time quantitative PCR was performed using the ABI PRISM 7000 Sequence Detector (Applied Biosystems, Foster City, CA, USA), with the qPCR mastermix (Eurogentec, Seraing, Belgium) and Taq- Man Gene expression assays (Applied Biosystems) for Ngb, Cygb and HIF-1a. The parameters for PCR amplification were 50 °C for 2 min, 95 °C for 10 min, and by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Each PCR reaction, including the nontemplate control, was performed in tripli- cate. VEGF and Mb were used as positive control genes. Several housekeeping genes were tested to choose the best internal control for each tissue (hydroxymethylbilane syn- thase for brain, eyes and skeletal muscle; small-subunit RNA for liver; and TATA box-binding protein for heart). The relative expression of mRNA was calculated using the comparative threshold cycle method. ROS detection The Amplex Red Hydrogen peroxide Assay kit (Molecular Probes, Eugene, OR, USA) was used to detect H 2 O 2 . In the presence of horseradish peroxidase, the Amplex Red reagent reacts with H 2 O 2 to produce highly fluorescent resorufin. Tissues were washed with ice-cold NaCl ⁄ P i and homogenized in extraction buffer. After two centrifugation steps (10 min 1000 g, 10 min 9000 g)at4°C on a Biofuge 13 centrifuge with 3743 rotor (Heraeus Instruments, Niedersachsen, Ger- many) 50 lg of total protein was added to a reaction mixture that contained 50 lm Amplex Red and 1 UÆmL )1 horserad- ish peroxidase. The excitation ⁄ emission wavelength filters were 544 nm ⁄ 590 nm. Each experimental condition was run in duplicate, and the mean ± SEM was calculated. Fluores- cence changes from the cells were converted to pmol released H 2 O 2 using a standard curve (0–200 pmol). Protein extraction and western blotting Tissues were crushed in liquid nitrogen and dissolved in lysis buffer [100 mm NaCl, 1 mm EDTA, 10 mm Tris ⁄ HCl, pH 7.5 plus protease inhibitor Complete (Roche Diagnos- tics, Basel, Switzerland)]. Samples were homogenized and sonicated. Samples (20 lg) were run on a 12.5% polyacryla- mide gel and electroblotted onto an Immobilon P membrane (Millipore, Billerica, MA, USA). Membranes were blocked in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and 5% nonfat dry milk for 1 h. After blocking, membranes were probed overnight at 4 °C with primary antibody [1 ⁄ 1000, Ngb and Cygb polyclonal antibodies [2]; 1 ⁄ 10 000, monoclonal anti-b-actin (Sigma-Aldrich, St Louis, MO, USA); 1 ⁄ 1000, polyclonal cleaved caspase-3 (Cell Signaling, Danvers, MA, USA)] in antibody dilution buffer (TBS-T containing 1% nonfat dry milk). Membranes were washed with antibody dilution buffer and incubated with horserad- ish peroxidase-labeled secondary antibody (1 ⁄ 2000; Dako, Copenhagen, Denmark) for 1 h at room temperature. After washing, antibody detection was accomplished with Super- signal West Femto maximum sensitivity substrate (Pierce, Rockford, IL, USA). The cleaved caspase-3 antibody detects endogenous levels of the large fragment (17 ⁄ 19 kDa) of acti- vated caspase-3 resulting from cleavage adjacent to Asp175. The antibody does not recognize full-length caspase-3 or other cleaved caspases. Signals were detected using a Lumi- Imager (Roche Diagnostics). image quant (Molecular Dynamics, Amersham Biosciences, NJ, USA) was used for quantitation, using b-actin for normalization. Statistics Statistical analysis of data utilized anova with least signifi- cant difference and Bonferroni post hoc tests (P £ 0.05). Acknowledgements We thank unknown referees for valuable comments. We would like to thank K. Brys and Professor Dr J. Vanfleteren (University of Ghent) for help with the ROS detection experiments. Professor Dr P. D’Haese (University of Antwerp) is acknowledged for giving us the opportunity to work on the ABI Prism 7000 Sequence detector. Dr B. M. y. Keenoy (University of Antwerp) is acknowledged for giving us the opportunity to work on the Fluoroskan Ascent Microplate Fluorim- eter. We wish to thank E. Geuens for helping us with the antibodies. This study was supported by a grant from EU (QLG-CT-2002-01548, ‘Neuroglobin and the survival of the neuron’) to LM. 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