Exendin-4 increases extracellular superoxide dismutase expression in cultured astrocytes

Received: April 02, 2017; Accepted: April 24, 2017; Published: April 26, 2017 Abbreviations: AD: Alzheimer’s disease; CNS: central nervous system; CRE: cyclic AMP response element; CREB: CRE binding protein; DMEM: Dulbecco’s modified Eagle medium; DTPA: diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid; Ex: exendin; FBS: fetal bovine serum; GLP-1: glucagon-like peptide-1; GSH: glutathione; HKR: HEPES-buffered Krebs Ringer solution; HPX: hypoxanthine; HRP: horseradish peroxidase; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-tetrazolium bromide; NBT: nitrotetrazolium blue chloride; NF-κB: nuclear factor-kappa B; O2 –: superoxide anion; PBS: phosphatebuffered saline; PD: Parkinson’s disease; ROS: reactive oxygen species; RT-PCR: reverse transcription polymerase chain reaction; SOD: superoxide dismutase; WST-1: water soluble tetrazolium-1; XOD: xanthine oxidase.

It was reported that Ex-4 permeated blood brain barrier [15]. In model mice of diabetes mellitus, Ex-4 injection could protect neurons through upregulation of glutamate transporters and neurotrophic factors [16,17]. In rodent models of brain ischemia, AD, and PD, Ex-4 could protect neurons from oxidative stress-induced impairment [18][19][20][21]. It was known that hyperglycemia in diabetes mellitus might elevate oxidative stress and induce mitochondrial impairment to cause neuronal toxicity [22,23]. Also in human ischemia, AD and PD, oxidative stress and metabolic disturbance might cause neuronal dysfunction [24][25][26].

Preparation of astrocytes culture
This study was carried out in compliance with the Guideline for Animal Experimentation at Osaka Prefecture University, with an effort to minimize the number of animals used and their suffering. Astrocytes were prepared as described previously [51]. In brief, cortex from 20-dayold embryos, which were taken out from pregnant Wistar rats deeply anesthetized, were cleared of meninges, cut into about 1 mm 3 blocks, and treated with 0.25% trypsin in Ca 2+ , Mg 2+ -free phosphate-buffered saline containing 5.5 mM glucose for 20 min at 37ºC with gentle shaking. An equal volume of horse serum supplemented with 0.1 mg/ ml of DNase I was added to the medium to inactivate the trypsin. Then, the tissues were centrifuged at 350 x g for 5 min. The tissue sediments were triturated through a pipette with DMEM containing 10% fetal bovine serum, 100 µg/ml streptomycin and 50 unit/ml penicillin. After filtering cell suspensions through a lens-cleaning paper (Fujifilm Co., Tokyo, Japan), the cells were plated on polyethyleneimine-coated 100 mm-diameter plastic dishes (Iwaki, Asahi Glass Co., Tokyo, Japan) at a density of 0.8-1.3 x 10 5 cells/cm 2 . Cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37ºC with changing medium every 3 days. After one week, astrocytes were replated to remove neurons. On days 12-14, they were replated onto 96-well plates (MS-8096F; for tissue culture, Sumitomo, Tokyo, Japan), 12-well plates (Iwaki), 35 mm-diameter plastic dishes (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.), or 60 mm-diameter plastic dishes (Thermo Fisher Scientific) using an ordinary trypsin-treatment technique at a density of 1.2 x 10 5 cells/cm 2 and stabilized for 1 day, then we used for experiments.
More than 90% of the cells were immunoreactively positive to glial fibrillary acidic protein (GFAP) using the antibody (Sigma). Less than 10% of the cells were positive to Iba-1 using the antibody (Wako).

Cell viability
To evaluate cell viability, we measured total mitochondrial activity with so-called MTT assay. In brief, after the cells were stimulated, the medium was changed with a fresh one and one-tenth volume of 5 mg/ml MTT solution was added. The cells were incubated for 1 h at 37ºC and the formazan generated by total mitochondrial activity was dissolved in dimethylsulfoxide, and then the color development was measured at 585 nm with a microplate reader (ARVO 1420 Multilabel counter, Wallac, Turuk, Finland). When we observed cell morphology under a phase-contrast microscope, the remaining cell number is almost consistent with the results of MTT assay.

Reverse transcription-polymerase chain reaction (RT-PCR)
Cultured astrocytes were washed with PBS, followed by extraction of mRNA using FavorPrep TM Tissue Total RNA Purification Mini Kit and subsequent synthesis of complementary DNA with oligo dT Superoxide dismutase (SOD) is a unique enzyme that converts O 2 into H 2 O 2 and O 2 by dismutating reaction [27] and known to be classified into three isozymes: cytosolic SOD (Cyt-SOD, Cu/Zn-SOD or SOD1) that contains Cu 2+ and Zn 2+ as enzyme cofactors, mitochondrial SOD (Mt-SOD, Mn-SOD or SOD2) in its matrix that contains Mn 2+ , and extracellular SOD (EC-SOD or SOD3) that contains Cu 2+ and Zn 2+ and has the enzymatic activity in extracellular space.
In the central nervous system (CNS), SOD isozymes are expressed and there are many reports suggesting that SOD plays important roles in defense against hypoxia-induced brain injury and neurodegenerative diseases such as PD [28][29][30][31][32][33][34][35]. However, most of these reports regard Cyt-SOD; not so much information on EC-SOD is known in CNS.
Although the expression level of EC-SOD in brain is considered to be much less than blood vessel or lung [36], it is reported that a proteolytic product of EC-SOD at C-terminal region is secreted in cerebrospinal fluid [37] and EC-SOD mRNA expression level increases in the brain after ischemia [38]. Furthermore, there are reports that mice overexpressing EC-SOD shows the increased resistance to focal cerebral ischemia [39,40], conversely EC-SOD deficiency in mice worsens outcome from focal cerebral ischemia [41]. Further, the beneficial function of EC-SOD is demonstrated in hyperoxia-induced brain injury in neonatal mice [42]. EC-SOD should play crucial roles in brain.
In addition, we previously reported that the expression level of EC-SOD in cultured astrocytes was higher than neurons and microglia by semi-quantitative RT-PCR and that lipopolysaccharide-stimulation increased SOD activity in the medium [43]. We also previously reported that dopamine incorporated into the cells through dopamine transporter triggered the EC-SOD induction via nuclear factor-kappa B (NF-κB) activation in cultured astrocytes [44]. Astrocytes play various important roles in CNS, such as maintenance of blood brain barrier, scavenging some neurotransmitters, control of ionic balance in brain parenchyma [45][46][47]. These functions of astrocytes serve the maintenance of brain homeostasis. Furthermore, it is reported that astrocytes protect endothelia from oxidative stress [48] and supply glutathione (GSH) to neurons and that astrocytes regulate the metabolism of ascorbic acid to protect neurons [49,50].
In the present study, we examined the effects of Ex-4 on EC-SOD expression and activity in cultured rat brain astrocytes. We found that exposure to Ex-4 increased the EC-SOD expressions in mRNA and protein, and SOD activities on the cell-surface and in the medium were not significantly affected by Ex-4. primers, mixture of dNTP (deoxyribonucleotide triphosphate), RNase inhibitor, Buffer RT and Omniscript Reverse Transcriptase (Omniscript Reverse Transcription Kit; Qiagen). Reverse transcriptase reaction was run at 37ºC for 60 min, followed by inactivation of the enzyme at 94ºC for 5 min, and an aliquot of synthesized complementary DNA was used for Realtime PCR.

Materials
Realtime PCR was performed in buffer containing SYBR ® Green Realtime PCR Master Mix (Toyobo) and each primer for the corresponding EC-SOD and 18S rRNA. PCR was performed with primers specific for each EC-SOD and 18S rRNA described below. The conditions of each PCR cycles for these primers were as follows: denaturation at 95ºC for 15 sec; annealing at 60ºC for 15 sec; and extension at 60ºC for 30 sec. The results were analyzed using Realtime PCR System (StepOne™; Applied Biosystems).

Western blotting
Cultured astrocytes were homogenized in 20 mM Tris-HCl (pH 7.5) buffer containing 1 mM EDTA and protease inhibitor cocktail (Sigma P8340). Each homogenate was added at a volume ratio of 4:1 to 50 mM Tris-HCl buffer (pH 6.8) containing 50% glycerol, 10% sodium dodecyl sulfate, 0.05% bromophenol blue and 25% 2-mercaptoethanol, followed by mixing and boiling at 100ºC for 5 min. Each aliquot in a certain amount of protein was loaded on a 10% polyacrylamide gel for electrophoresis at a constant voltage of 120 V for 2 h at room temperature and subsequent blotting to a polyvinylidene fluoride membrane previously treated with 100% methanol. After blocking by 5% skimmed milk dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 137 mM NaCl and 0.05% Tween 20, the membrane was reacted with antibodies against EC-SOD or β-actin followed by a reaction with anti-rabbit or anti-mouse IgG antibody conjugated with peroxidase. Proteins reactive with those antibodies were detected with the aid of chemiluminescence detection reagents (Immobilon™ Western HRP Substrate; Millipore Corporation, Billerica, MA, U.S.A.) and analyzed with lumino-image-analyzer (LAS-4000, Fujifilm). The graphs showed EC-SOD/β-actin ratio of the density of detection bands.
Protein concentrations were determined by the method of Bradford using CBB color solution (Nacalai Tesque, Kyoto, Japan), according to the manufacturer's protocol, with bovine serum albumin as the standard.

Measurement of SOD activity on cell surface
SOD activity on cell surface was measured by the assay as described previously [43]. After the cells were rinsed with HEPES-buffered Krebs Ringer solution (HKR; 130 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO 4 , 50 mM HEPES, 1 mM NaH 2 PO 4 , 5.6 mM glucose, 1.8 mM CaCl 2 , pH 7.4) twice, 90 µl of 0.3 mM HPX, 0.3 mM DTPA and 33 µM WST-1 in HKR was added into each well. Then the reaction was started with the addition of 20 µl of 46 mU/ml XOD and the absorbance (Abs) at 450 nm was immediately measured as blank, using a microplate reader. The plate was incubated in a CO 2 incubator for 30 min, and then the absorbance was measured. For standard, various concentrations of SOD enzyme (Sigma S-8160 from bovine liver) were added into cell-free wells in the same plate; then, the absorbance was measured simultaneously.
The standard curve was established as follows. The absorbance at 30 min was used; at first, the blank values (time 0) were subtracted. The difference between each value and 0 SOD was plotted against the standard SOD concentrations: the central linear part of the sigmoid curve was used for the calculation of SOD activity. This procedure is based on the inhibition of O 2 detection with an artificial O 2 generator; therefore, the procedure is not applicable when the cells themselves have O 2 generating activity. We examined the color development of WST-1 added on the astrocytes without addition of XOD: the absorbance change was less than 0.002 Abs/h and revealed that astrocytes did generate almost no O 2 -.

Measurement of SOD activity in medium
We had attempted to measure the SOD activity in the medium released from the cell similarly as described above; however, the measurement was disturbed by some components of the DMEM. Therefore, in order to measure the SOD activity released into the medium, we used HKR for the drug-stimulation reaction instead of DMEM. It is supplemented that cell viability in HKR was not different from that in DMEM with and without LPS (data not shown).

Data analysis
For statistical analysis of the data, one-way ANOVA followed by Tukey-Kramer multiple comparison procedure or Student's t-test was used. Differences between treatments were considered statistically significant when p < 0.05.

Expression of EC-SOD mRNA increased in astrocytes after Ex-4 stimulation
Cultured astrocytes were stimulated with 10 nM Ex-4 for indicated time, and then the mRNA expressions of EC-SOD were analyzed by realtime RT-PCR. The expression of EC-SOD mRNA was significantly increased by 10 nM Ex-4 for 3 h exposure, it was remarkably increased at 6 h and the increased level was sustained up to 24 h (Figure 1).

Effect of Ex-4 and/or exendin (9-39) on the EC-SOD protein
In addition to the mRNA level, we examined the level of EC-SOD protein. The cells were stimulated with various concentrations of Ex-4 for 24 h, and then the expression of EC-SOD protein was assessed by western blotting. The expression level of EC-SOD protein increased in a dose-dependent manner and with 10 nM Ex-4, significantly (Figure 2A).
The cells were preincubated with 100 nM exendin , an antagonist of GLP-1 receptor, for 30 min and stimulated by 10 nM Ex-4 for 24 h, and then the expression of EC-SOD protein was assessed by western blotting. Preincubation with 100 nM exendin (9-39) inhibited Ex-4-increased EC-SOD expression ( Figure 2B).

Effect of Ex-4 on cell-surface SOD activity and the cell viability
We examined the effect of Ex-4 on cell-surface SOD activity in cultured astrocytes. The cells were incubated with various concentrations of Ex-4 for 24 h, and washed with HKR. Then, SOD activity on the cell-surface was assayed. The activity was not significantly affected ( Figure 3A).
We also examined the effect of Ex-4 on cell viability in cultured astrocytes. The cells were incubated with various concentrations of Ex-4 for 24 h, and cell viability was assessed by MTT assay. Cell viability was not affected by Ex-4 used in the present study ( Figure 3B).

Effects of Ex-4 and/or exendin (9-39) on SOD activity in the medium
We examined the effect of Ex-4 on SOD activity in the medium. Cultured astrocytes were preincubated with 100 nM exendin (9-39) for 30 min and stimulated by 10 nM Ex-4 for 24 h in HKR, and then SOD activity in the medium was assayed. The activity was affected by neither Ex-4 nor exendin (9-39) (Figure 4).

Discussion
In the present study, we demonstrated that the expressions of EC-SOD, both of mRNA and protein, were induced when the cells were stimulated by Ex-4 in cultured rat cortex astrocytes. However, the activities of SOD on the cell-surface and in the medium were not significantly affected by Ex-4. The Ex-4-increased EC-SOD expression was inhibited by an antagonist of GLP-1 receptor. These results suggest that Ex-4 increases the expressions of EC-SOD mRNA and protein via binding to GLP-1 receptor.
In cultured astrocytes, 10 nM Ex-4 increased EC-SOD mRNA time-dependently for 1-24 h, and 1-10 nM Ex-4 exposure for 24 h increased EC-SOD protein in a dose-dependent manner, significantly with 10 nM. The increased expression of EC-SOD protein by Ex-4 was blocked by exendin , an antagonist of GLP-1 receptor. GLP-1 receptor is a G-protein coupled receptor, and binding of agonists to it causes activation of adenylate cyclase to increase cAMP and activate protein kinase A [52]. Ex-4 treatment reported to induce the elevation of intracellular cAMP levels in astrocytes [52]. The promoter region of EC-SOD contains various regulatory elements including cAMP response element (CRE) [53]. Therefore, Ex-4 in the present study might increase EC-SOD mRNA via intracellular cAMP production. Moreover, the promoter region of EC-SOD contains various regulatory elements other than CRE, including antioxidant response element, activator protein-1 binding sites, NF-κB motifs, and xenobiotic response elements [53]. Further investigation is necessary to elucidate the total mechanisms of the EC-SOD induction by Ex-4 stimulation,   inclusive of other transcriptional factors as well as CRE-binding protein (CREB).
It was reported that Ex-4 administration protected neurons through the upregulation of antioxidants in ischemic rat brain [18]. However, in the present study, the activities of SOD on the cell-surface and in the medium were not significantly affected by Ex-4 treatment for 24 h. The effects of Ex-4 on mRNA and protein only in EC-SOD among three isozymes of SOD were assessed in the present study; therefore, the effects of Ex-4 on the expressions of other two SODs, Cu/Zn-SOD and Mn-SOD should be further investigated. Moreover, it is likely that Ex-4 might affect the utilization efficiency of Cu 2+ and Zn 2+ which are contained as enzyme cofactors and needed for enzymatic activity of EC-SOD.
EC-SOD is a unique enzyme having the enzymatic activity in extracellular space and at least two steps are known to be occurred for secretion [27,54]. It was reported that N-glycosylation was essential for the transport of intracellular EC-SOD protein to cellular membrane [55]. Further, EC-SOD is known to be released through the proteolytic processing at binding region in various cells including glial cell line U1169 CG [56]. The C-terminal proteolytic processing of EC-SOD was reported to be regulated by redox state [57]. In the present study, these post-translational modifications might not be stimulated by Ex-4 treatment in cultured astrocytes although Ex-4 increased the expressions of EC-SOD mRNA and protein. Further investigation is needed to elucidate the effects of Ex-4 on SOD activity in more detail; however, the induced EC-SOD in astrocytes by Ex-4 exposure might play a role in neuronal protection.

Conclusion
In the present study, Ex-4 triggered the EC-SOD induction via binding to GLP-1 receptor in cultured astrocytes. EC-SOD induced by Ex-4 in astrocytes may play a role in extracellular anti-oxidative defense for surrounding neurons. The regulation of EC-SOD expression in astrocytes should be an important target in treatment of CNS diseases.