Neuroscience Letters 444 (2008) 106–108
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Heme oxygenase expression and activity in immortalized hypothalamic neurons GT1–7 Nadia Mores a , Stefania Errico a , Angela Pusateri b , Eugenio Barone a , Cesare Mancuso a,∗ a b
Institute of Pharmacology, Catholic University School of Medicine, Largo Francesco Vito 1, 00168 Roma, Italy Institute of Anaestehesiology, Catholic University School of Medicine, Largo Francesco Vito 1, 00168 Roma, Italy
a r t i c l e
i n f o
Article history: Received 3 July 2008 Received in revised form 1 August 2008 Accepted 6 August 2008 Keywords: Bilirubin Carbon monoxide Heme oxygenase Hypothalamic neurons Gonadotropin releasing hormone GT1–7 cells
a b s t r a c t Heme oxygenase (HO), the main enzyme deputed to heme metabolism, has been identified as two main isoforms called HO-1 and HO-2. HO-1 is inducible and plays a main role in the cellular oxidant/antioxidant balance whereas HO-2 is constitutive and involved in the physiological metabolism of heme. However, it is noteworthy to mention that HO contribute to the regulation of the hypothalamic release of neuropeptides such as corticotrophin-releasing hormone and arginine-vasopressin and could modulate the pulsatile release of gonadotropin releasing hormone (GnRH). GT1–7 cells are immortalized hypothalamic neurons and a valuable tool to evaluate hypothalamic neuroendocrine control of reproduction. The aim of this work was to investigate and characterize the presence of HO isoforms in the GT1–7 hypothalamic neurons. Hemin, a well-known inducer of HO-1, significantly increased HO activity, whereas dexamethasone did not modify HO-2 activity. Moreover, hemin and DEX, in combination, did not have any additive effect on HO activity in GT1–7 neurons. Furthermore, basal HO-1 immunoreactivity identified in GT1–7 cells, was significantly up-regulated by hemin. Conversely, no HO-2 immunoreactivity was detected. Taken together, these results suggest the presence of functional HO-1 in GT1–7 immortalized hypothalamic neurons and open new avenues about the use of this cell line for the study of HO modulation of GnRH secretion and reproduction. © 2008 Elsevier Ireland Ltd. All rights reserved.
Heme oxygenase (HO) is a microsomal enzyme and catalyzes the oxidation of the alpha-meso-carbon bridge of heme moieties resulting in the generation of ferrous iron, carbon monoxide (CO) and biliverdin [12,19,20]. This latter is then reduced by the cytosolic biliverdin reductase into bilirubin (BR), a molecule endowed with antioxidant and antinitrosative activities [12,14,15,17,25]. Two isoforms of HO have been identified and named as HO-1 and HO-2 [12,20]. These isoforms catalyze the same reaction, but they play a different role in the regulation of cell homeostasis [11]. In fact, HO-1 is inducible under conditions of oxidative and nitrosative stress and is involved in the early phase of the cellular stress response, whereas HO-2, constitutive in nature but inducible by few selected stimuli including steroid hormones [24], is responsible for the physiologic degradation of heme and serves as an endogenous sensor of cellular oxygen, CO and nitric oxide [1,2,11–13]. HO-1 is ubiquitary, but particularly abundant in liver and spleen, whereas HO-2 is localized in specific organs such as brain, kidney and testis [12,20]. The central nervous system, is endowed with
∗ Corresponding author. Tel.: +39 06 30154367; fax: +39 06 3050159. E-mail address:
[email protected] (C. Mancuso). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.016
very high HO activity under basal conditions, mostly accounted for HO-2, the latter being expressed in neuronal populations in forebrain, hippocampus, hypothalamus (supraoptic nucleus), midbrain, basal ganglia, thalamus, cerebellum and brainstem [12,20]. On the contrary, HO-1 is present in very small amounts and is localized in sparse groups of neurons, including the ventromedial (VM) nucleus and paraventricular nucleus (PVN) of the hypothalamus [12,20]. These findings indicate that the activation of both HO-1 and HO-2 can be induced by many stimuli within areas that are primarily involved in the central regulation of the stress response and reproduction. Indeed, neurons located within the parvicellular part of the PVN release both corticotrophin-releasing hormone (CRH) and arginine-vasopressin (AVP), the neuropeptides that initiate the endocrine response to stressors stimulating the release of pituitary ACTH [20]. In the medial preoptic area gonadotropin releasing hormone (GnRH) is produced by a small number of specialized neurosecretory cells [27] and controls the reproduction via the episodic secretion of pituitary gonadotropins [6]. HO-1 has been also found in glial cells, where its gene expression can be induced by oxidative stress [12,20]. In 1997, Mahin Maines described a third HO isoform called HO-3 [21]. However, the regulation of HO-3 gene expression is poorly understood and its possible role in physiology and pathology remains to be further clarified.
N. Mores et al. / Neuroscience Letters 444 (2008) 106–108
Previous studies have shown the importance of HO and its gaseous product CO in the regulation of hypothalamic functions such as the release of neuropeptides [4,16,18,23]. HO and CO have been demonstrated to reduce the hypothalamic release of both CRH and AVP stimulated by depolarizing and immuno-inflammatory stimuli in the rat [16,18,23]. Furthermore, hemin has been shown to increase the basal GnRH release in the rat hypothalamus [10]. The aim of this work was to investigate and characterize the presence of HO isoforms in the GT1–7 cell line. GT1–7 cells are immortalized hypothalamic neurons exhibiting spontaneous electrical activity and pulsatile GnRH release at a frequency similar to that of GnRH secretion in vitro [8]. GT1–7 cells express among others functional type 2 CRH receptor and represent a valuable tool to investigate the hypothalamic crosstalk in neuroendocrine function [5,7,22]. GT1–7 cells, kindly provided by Dr. Pamela L. Mellon (University of California, San Diego) [22], were cultured in culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 0.584 g/l l-glutamate and 4.5 g/l glucose, mixed 1:1 with F-12 medium (Invitrogen) containing 0.146 g/l l-glutamine, 1.8 g/l glucose, 100 g/ml gentamicin, 2.5 g/l sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (Invitrogen). GT1–7 cells were plated in 12-well plates at a density of 106 cells/ml and maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 /95% air. All the experiments were performed in serum free DMEM/F12 (SFM) containing 0.1% bovine serum albumin (Sigma–Aldrich, Milan, Italy), 30 mg/l bacitracin (Sigma–Aldrich), and pH 7.4. Hemin (Sigma–Aldrich), was dissolved in 0.1 M NaOH at the stock solution of 10 mM and then diluted in SFM at the desired working concentration. Whole cell preparations were analyzed by SDS-PAGE and HO-1 immunoreactivity was determined by Western blot. GT1–7 cells were washed twice with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.4), collected with lysis buffer and stored at −80 ◦ C. For immunoblot analysis of HO-1, 30 g of total proteins was separated by SDS-gel electrophoresis on 12.5% acrylamide gels, and then transferred on polyvinylidene difluoride membrane of 0.45-m pore size. HO-1 immunoreactivity was detected by incubation of blots with a rabbit polyclonal first antibody (1:1000, Stressgen, Victoria, Canada) followed by peroxidase-coupled goat anti-rabbit IgG (H + L), and visualized by chemiluminescence (Invitrogen). The immunoreactive bands were analyzed as three-dimensional digitized images using a GS-700 Imaging Densitometer (Bio-Rad, Hercules, CA, USA). The optical density (OD) of images is expressed as volume (OD × area) adjusted for the background, which gives arbitrary units of adjusted volume. HO activity was evaluated by measuring the amount of BR released in the cell culture medium as described by Turcanu et al. [26]. LDH in cell free culture supernatant was determined using a Promega (Milan, Italy) fluorimetric enzimatic assay following the manufacturer’s instructions. Data are expressed as mean ± S.E.M. of (n) replicates per group. Differences were considered statistically significant if P < 0.05. The potential toxic effects of hemin on GT1–7 viability were firstly assessed in time and dose dependent experiments. Hemin, in the dose-range 1 nM to 1 M did not affect GT1–7 viability after 1–24 h of incubation as evaluated by LDH release (data not shown). Afterwards basal and stimulated HO activity was measured. As shown in Fig. 1, GT1–7 cells exhibited a basal HO activity, significantly up-regulated after 24 h exposure to the HO substrate/inducer, hemin (1 M). Conversely, dexamethasone (DEX, 1 g/ml for 24 h), which has been shown to induce HO-2 [11,12,24], did not show any effect on HO activity. However, there was no additive effect by hemin and DEX combined, at the previously effective concentrations and HO activity was quite similar to that observed after exposure to hemin alone (Fig. 1). Basal HO1 immuno-like reactivity was positively detected by Western blot
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Fig. 1. Heme oxygenase activity expressed as bilirubin levels in GT1–7 hypothalamic neurons. GT1–7 cells were incubated with hemin (H, 1 M) and dexamethasone (DEX, 1 g/ml) for 24 h in serum free DMEM/F12 medium (1/1, v/v) supplemented with 0.1% bovine serum albumin and 30 mg/l bacitracin, pH 7.4. At the end of incubation, culture medium was collected and the bilirubin concentration was measured as described by Turcanu et al. [26]. Data are expressed as pmol of bilirubin/106 cells, mean ± S.E.M. of six replicates per group. **P < 0.01 vs. Control (Ctrl).
analysis on GT1–7 neurons (Fig. 2). Moreover, exposure to hemin 1 M, increased HO-1 immunoreactivity as early as 12 h reaching the maximum and statistical significance at 24 h (Fig. 2). No effect was detected after shorter exposures (Fig. 2). Finally, no basal HO2 immunoreactivity has been detected in GT1–7 cells (data not shown). This last result was not surprising or unexpected. In fact, although HO-2 is considered the more abundant isoform in neuronal cells, several reports have demonstrated that hypothalamic regions in particular the PVN, contain neurons expressing mainly HO-1 which can be up-regulated under stressful conditions [3,12]. Accordingly, Lamar et al. have shown the lack of HO-2 mRNA in both the preoptic area and mediobasal hypothalamus, thus strengthening the hypothesis that HO-1 is the isoform principally involved in the regulation of hypothalamic neuroendocrine function [9]. This finding is in good agreement with the evidence of a slight increase in HO activity following DEX exposure of GT1–7 hypothalamic neurons (Fig. 1). In fact, the HO-2 gene contains, within the promoter, a glucocorticoid-responsive element responsible for its up-regulation [12,13], whereas HO-1 is not sensitive to glucocorticoids, but its overexpression is regulated by the interaction of several stimuli (reactive oxygen species, hemin, cytokines, etc.) with the promoter region known as antioxidant responsive element (ARE) mediated by the heterodimeric basic leucine zipper factors NF-E2-related factors 2 (Nrf2) and AP-1 [12,13]. Furthermore, the ability of hemin to stimulate HO activity in GT1–7 neurons after 24 h exposure resembled previous results obtained in rat hypothalamic explants after 20 min stimulus and provided new evidence for HO involvement in both rapid and long-term degradation of heme [16]. However, in the case of the short-term (20 min) incubation with hemin the up-regulation of hypothalamic HO-1 is quite unlikely and the increase in HO activity may be due to the relative abundance in its substrate; on the contrary, when GT1–7 neurons have been incubated with hemin 1 M for 24 h, the increased HO activity has been paralleled by a significant HO-1 up-regulation (Figs. 1 and 2). Although it is important to dissect the time-dependence of both HO activity and HO-1 up-regulation, the pathophysiologic implications of the final common effect, namely the degradation of heme, do not change. This last finding is particularly meaningful considering that heme is pro-oxidant if produced in excess and its catabolism generates both CO and BR, two molecules endowed with neuroendocrine and cytoprotective activities, respectively [17,20,25]. Taken together, these results suggest that the hypothalamic immortalized neurons GT1–7 represent not only a valuable tool to investigate the hypothalamic control of reproduction and release of neuropep-
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Fig. 2. Heme oxygenase-1 immunodetection in GT1–7 hypothalamic neurons. GT1–7 cells were incubated with hemin (H, 1 M) for 8, 12 and 24 h in SFM medium (1/1, v/v) supplemented with 0.1% bovine serum albumin and 30 mg/l bacitracin, pH 7.4. At the end of incubation, cells were collected, lysed and analyzed by Western blot using a rabbit polyclonal anti-heme oxygenase-1 antibody. Each experiment was performed in triplicate. Representative gels are shown. Bar graphs show the analysis of band optical intensity evaluated by adjusted volume. *P < 0.05 vs. Control (Ctrl); Std, standard.
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