Original Research Paper
Erythropoietin exerts cell protective effect by activating PI3K/Akt and MAPK pathways in C6 Cells Min-Soo Kwon1, Mi-Hee Kim2, Seon-Hee Kim2, Ki-Dae Park2, Si-Hyung Yoo2, Il-Ung Oh2, Suenie Pak2, Young-Jun Seo3 1
Department of Pharmacology, School of Medicine, CHA University, Yatap-dong, Bundang-gu, Seongnam, Gyeonggi-do, Republic of Korea, 2Advanced Therapy Products Research Division Pharmaceutical and Medical Device Research Department, NIFDS Korea Food and Drug Administration, Gangoe-myeon, Cheongwon-gun, Chungcheongbuk-do, Republic of Korea, 3Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Even though erythropoietin (EPO) is a neurotropic cytokine that is recognized widely for its role in the development, maintenance, protection, and repair of the nervous system, there are few reports concerning EPO-mediated influences on the glial cells in the central nervous system. In this study, we investigated antiinflammatory and anti-apoptotic effects of EPO on C6 glioma cells (C6 cells). Erythropoietin did not attenuate inflammatory response, such as nitrite production, iNOS gene expression, and pro-inflammatory cytokines when LPS/TNF-alpha mixture was treated. However, EPO increased C6 cell viability by exerting cell protective effect against staurosporine stimulation. Erythropoietin increased the transient Akt expression at 30 minutes and induced the gradual elevation of ERK1/2 and p38 expression as time progressed. The cell protective effect of EPO was also significantly attenuated with pretreatment of specific PI3K, pERK1/2, or pP38 inhibitor. In summary, these results suggest that EPO may exert its cell protective functions via the direct cell protective activity rather than via its anti-inflammatory effect. Moreover, the PI3K/Akt and mitogen activated protein kinase (MAPK) pathways may be responsible for cell survival against cytotoxicity. Keywords: Erythropoietin, C6 cells, Cell protection, Anti-inflammation, Signal pathway
Introduction Erythropoietin (EPO) has been originally characterized as a major hematopoietic growth factor, but recently it has been proposed to be one of the most efficient neuroprotective agents as well. Several in vitro/vivo studies have provided evidence for the critical role of EPO in the control of neuronal function and have suggested a potential neuroprotective value in CNS disorders with various pathophysiological origins, such as neurotoxic and excitotoxic stress,1,2 cerebral ischemia, hypoxia,3,4 trauma,5 and inflammatory and neurodegenerative diseases.6,7 Based on these experimental reports, EPO has undergone evaluations in clinical trials for patients with various neurological diseases, such as stroke, traumatic brain injury, and subarachnoid hemorrhage.8–10
Correspondence to: Young-jun Seo, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Email: [email protected]
ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132813Y.0000000284
Erythropoietin binding causes the EPO receptor (EPOR) to form a homodimeric or EPOR–beta common receptor (BCR) heterotrimeric structure that triggers complex intracellular signal cascades, such as JAK2/STAT5 signaling, phosphatidylinositol 3-kinase (PI3K) signaling, protein kinase C, mitogen activated protein kinase (MAPK), and nuclear factor (NF)-kB pathways.1,6,11,12 The EPOR stimulation in the target cells leads to the proliferation and differentiation of stem cells, the inhibition of apoptosis, and anti-inflammatory and anti-oxidative effects. In the nervous system, mechanisms including direct neuroprotection, anti-inflammation, anti-apoptotic, antioxidative effects, stimulation of neurogenesis, and modulation of neurotransmission have been suggested to be crucial bases underlying the beneficial effects of EPO.13,14,15 Astrocytes, the most prevalent glial cell types in the brain, provide metabolic/trophic support to neurons and modulate synapse. Therefore, impairment in these astrocyte functions can critically influence neuronal
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survival. Recent studies have demonstrated that apoptosis of astrocytes may contribute to pathogenesis of many acute or chronic neurodegenerative diseases, such as cerebral ischemia, Alzheimer’s disease, and Parkinson’s disease.16–19 In addition, astrocytes have high concentration of antioxidants, such as low mole>cular weight antioxidants (e.g., ascorbic acid and glutathione peroxidase) and antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), which protect the surrounding cells containing neurons from oxidant stress-induced cell death.20 As a model mimicking astrocyte properties, C6 glioma cell line has been used frequently.21 Additionally, later stages of C6 glioma cells exhibit several characteristics that resemble astrocytes. Therefore, C6 glioma cells have been used as an alternative cell model for astrocytes owing to its convenience for rapid screening.22 Even though other brain cells, such as neurons, oligodendrocytes, microglia, and endothelial cells can synthesize EPO in response to hypoxia-ischemia or other stressors, astrocytes are the main cellular source of local EPO.23,24 The local production of endogenous EPO may act in paracrine and autocrine manners to provide a neuroprotective effect. Since anti-oxidative effects are involved in the neuroprotective process of EPO in the endothelial cells, this raises the possibility that EPO may also play a role in cellular defenses against oxidative stress and subsequent apoptosis in astrocytes. In addition, the neuroprotective effects of EPO have been described in various models of neurotoxicity, but there is limited evidence regarding EPO-mediated influences on glial cells. Thus, we investigated the anti-apoptotic and anti-inflammatory effects of EPO against various toxic and inflammatory stimulations in cultured C6 glioma cells (C6 cells) mimicking astrocytes. We also examined the possible mechanisms of these effects.
Materials and Methods Cell culture and drug treatment Rat C6 glioma cells were obtained from the Korea Cell Line Bank and maintained in Dulbecco’s modified Eagle’s medium/F12 (Gibco, USA) containing 10% heat-inactivated fetal bovine serum (Gibco, USA), 2.2 g/l sodium bicarbonate, 0.6% (w/v) Dglucose, and 50 mg/ml penicillin and streptomycin. Cells were plated on 25-cm2 culture flasks (Corning, USA). The cultures were incubated at 37uC in 5% CO2 for 3 days. The cells were incubated with a serum-free culture medium for 24 hour prior to the incubation with drugs. Various EPO doses (0.0005–5 units/well) were co-treated with the LPS/TNF mixture at the same time or were pretreated 24 hours prior with the LPS/TNF mixture (50/5 mg/well) in 96 well plates.
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Each experiment was repeated for at least three different cultures.
Immunofluorescence C6 cells were plated onto cover slips that were precoated with collagen (Sigma-Aldrich, St Louis, MO, USA) in DMEM containing 10% FBS and P/S and were incubated at 37uC in humidified 5% CO2. After incubation, the cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 minutes at room temperature. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% and blocked with 10% serum for 60 minutes at 24uC. Samples were incubated with a primary antibody (1:100) for 16 hours at 4uC. Fixed cells were preincubated in 10% normal goat serum for 30 minutes to block nonspecific staining. For the staining of EPOR, the cells were incubated with rabbit antibodies against EPOR (abcam #ab61162, USA) for a minimum of 1 hour at room temperature. After washing with PBS, they were stained with fluorescein isothiocyanate-labeled antibodies against mouse IgG or Texas red-labeled antibodies against rabbit IgG and coverslipped with VECTASHIELD Mounting Media with DAPI. All specimens were observed under a confocal laser-scanning microscope (Olympus LX70 fluorescent microscope equipped with a HXP 120 C Hg lamp and DAPI and RFP filter-set).
Assay for NO synthesis NO synthesis was determined by an assay that detects nitrite, a stable reaction product of NO with molecular oxygen, in culture supernatant. Briefly, 100 ml of culture supernatant was allowed to react with 100 ml of Griess reagent25 and incubated at room temperature for 15 minutes. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture media served as blanks in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay.
Isolation of total RNA Total cellular RNA was extracted from C6 glioma cells using a rapid guanidine thiocyanate–water-saturated phenol/chloroform extraction procedure and subsequent precipitation with acidic sodium acetate.26 Total cellular RNA in the aqueous phase was precipitated with ice-cold isopropyl alcohol. Isolated RNA samples were subjected to spectrophotometric analysis at 260 and 280 nm. The separated organic layer was extracted twice with an equal volume of sterilized water (Millipore, Billerica, MA, US). Adding two volumes of absolute ethanol to the water-extracted organic phase precipitated the proteins. The RNA pellets were washed twice with cold absolute ethanol and dried. The dried pellets were
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dissolved in a denaturing buffer (6 M guanidinium chloride, 20 mM Tris–HCl [pH 8.0], and 1 mM EDTA). The RNA samples were dialyzed against a renaturing buffer (20 mM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 0.4 mM phenylmethylsulfonyl fluoride, and 20% glycerol) at 4uC. The concentration of RNA was determined with the Coomassie Blue protein assay reagent (Pierce Chemical Co., Rockford, IL, USA) using bovine serum albumin (BSA) as the standard.
Real-time PCR analysis The expression of TNF-alpha, IL-1beta, iNOS and GAPDH mRNA was evaluated by real-time PCR using QuantiTectTM SYBRH Green PCR Kit (Qiagen, Germany). All PCRs were performed in a total volume of 20 ml using the QuantiTectTM SYBR Green PCR Kit (Qiagen, Germany). Each reaction contained 1.5 ml of cDNA, 6.5 ml RNasefree water, 1 ml each of sense and antisense primers (20 mM), and 10 ml of 26 SYBR Green PCR Master Mix (containing QuantiTect SYBR Green PCR buffer, dNTPs, SYBR Green I dye, ROX dye, and HotStarTaq DNA polymerase). After an initial denaturation step at 95uC for 30 seconds, temperature cycling with a total of 40 cycles was initiated. Each cycle consisted of a denaturation phase at 95uC for 30 seconds, an annealing phase at 60uC for 30 seconds, and an elongation phase at 72uC for 30 seconds. Amplification was followed by a melting curve analysis to verify the correctness of the amplification. A negative control with water instead of cDNA was run within every PCR to assess the specificity of the reaction. To verify the accuracy of the amplification, PCR products were further analyzed on ethidium bromide-stained 2% agarose gel. For data analysis, Rotor-Gene 6000 Series Software 1.7 (Build 87) was used. Results are given as a ratio of the amount of TNF-alpha, IL-1beta, iNOS mRNA to that of GAPDH mRNA. The following primers were used: TNF-alpha (NM 012675) sense: AAC TCG AGT GAC AAG CCC GTA G, antisense: GTA CCA CCA GTT GGT TGT CTT TGA; IL-1beta (NM 031512) sense: GCT GTG GCA GCT ACC TAT GTC TTG, antisense: AGG TCG TCA TCA TCC CAC GAG; iNOS (NM 012611) sense: CTG TGT GCC TGG AGG TTC TAG ATG, antisense: AAG TAG GTG AGG GCT TGC CTG A; and GAPDH (NM 017008) sense: GGC ACA GTC AAG GCT GAG AAT G, antisense: ATG GTG GTG AAG ACG CCA GTA. The entire process to RTPCR was conducted three times independently. Each real-time PCR result (independently three times) was quantified with GraphPad Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA, USA) for statistical analysis.
Cell protective effect of erythropoietin
Western blot analysis The cells were washed with ice-cold Tris-buffered saline (TBS; 20 mM Trizma base and 137 mM NaCl, pH 7.5) and lysed in 16SDS sample loading buffer (62.5 mM Trizma base, 2% w/v SDS, 10% glycerol); after sonication and centrifugation at 15 0006g for 5 minutes, the supernatant was used for western blot assay. The protein concentrations of the samples were determined with a detergent-compatible protein assay reagent (Bio-Rad, Hercules, CA, USA), using BSA as standard. Samples were boiled for 3 minutes with 0.1 volume of 10% beta-mercaptoethanol and 0.5% bromophenol blue mix. Of the total cellular protein, 50 mg was resolved by electrophoresis on 8% polyacrylamide gels, electro-transferred to a PVDF membrane (Amersham Pharmacia, Buckinghamshire, UK) and blocked with TBST (10 mM Trizma base (pH 7.4), 150 mM NaCl, and 1% Tween 20) with 5% skim milk. After incubation with antiserum against pAkt, pERK, pP38, pJNK, Akt, and betaactin (#9271, #9102, #9211, #9251, #4691, #4967; Cell Signal Technology, CA, USA) for 2 hours at room temperature, the membranes were washed with TBST three times and then incubated with horseradish peroxidase conjugated anti-rabbit IgG for 1 hour. The membranes were visualized by using ECL-plus (Amersham Pharmacia) after washing with TBST buffer.
LDH assay LDH leakage from damaged cells was measured using a Roche LDA assay kit that quantitatively measures LDH, a stable cytosolic enzyme that is released upon cell lysis.27 The cells were treated with EPO 24 hours after seeding (1 unit/well) and incubated for another 24 hours Then, 50 ml of cell culture medium was collected from each well and plated on a new microtiter plate. Next, 50 ml of staurosporine (0.2 mM) was added to the wells, and the plates were incubated for 30 minutes at room temperature. The optical density at 490 nm was measured with a standard microplate reader. Each experiment was performed in triplicate. The LDH leakage (%) relative to the control wells containing cell culture medium without EPO and staurosporine, was calculated as [test][control] 100, where [test] was the absorbance of the test sample and [control] was the absorbance of the control sample
Drugs SB203580, PD98059, wortmannin and LY294002 were purchased from Cellsignal Technology (#5633, #9900, #9951, 9901; USA). Staurosporine was purchased from Roche Applied Science, IN, USA. SB203580, PD98059, wortmannin, LY294002, and staurosporine were dissolved in 1% DMSO solution that was diluted with the DMEM/F12 media.
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Figure 1 The expression of erythropoietin receptor (EPOR) in C6 cells. Immunofluorescence staining of C6 cells with an anti-EPOR antibody was performed. The EPOR was well expressed in the C6 cells derived from rat astrocytes.
LY294002 (50 mM) and wortmannin (1 mM) as PI3K/ pAkt inhibitors, SB203580 (10 mM, pP38 inhibitor), or PD98059 (50 mM, pERK inhibitor) was pretreated 1 hour prior to the EPO treatment. Drug doses of SB203580, PD98059, wortmannin, and LY294002, which were determined as critical concentrations for inhibition, were chosen based on the product reference data and previously published research. The controls were pretreated with 20% DMSO solution that was diluted with the DMEM/F12 media.
Statistical Analysis Data were presented as mean¡SEM. The statistical significance of differences between groups was assessed with one-way ANOVA with Bonferroni’s post hoc test or t-tests (and nonparametic tests) using GraphPad Prism version 5.00 for Mac OS X, (GraphPad Software, San Diego California USA, www.graphpad.com); P , 0.05 was considered significant.
Results Erythropoietin receptor expression in C6 cells To evaluate EPOR expression in C6 cells, we performed the confocal study. As shown in Fig. 1, EPOR expression was confirmed in C6 cells.
Effect of EPO on nitrite production increased by LPS/TNF mixture in C6 cells To observe whether EPO has an inhibitory effect on nitrite production in C6 cells with LPS/TNF mixture, we pretreated or co-treated EPO in C6 cells with LPS/ TNF, and then nitrite assay was performed. As the effect of EPO may depend on the concentration and treatment time in the C6 cell with the LPS/TNF mixture, we pretreated or co-treated the EPO in various doses. As shown in Fig. 2, pre or co-treated EPO did not have any effect on the increase in nitrite concentration in C6 cells with the LPS/TNF mixture regardless of its concentration (0.0005–5 units/well).
Effect of EPO on iNOS, TNF-alpha, and IL-beta mRNA expression induced by LPS/TNF mixture in C6 cells To observe the anti-inflammatory effect of EPO on mRNA levels of pro-inflammatory cytokines, we pretreated a vehicle (20% DMSO in media) or EPO in C6 cells 24 hours prior to the LPS/TNF treatment. The iNOS, TNF-alpha, and IL-1beta mRNA level
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were examined over several time points (30 minutes, 1 hour, 6 hours, 24 hours). As shown in Fig. 3, iNOS, TNF-alpha, and IL-1beta showed a peak level 6 hours after the LPS/TNF mixture treatment. However, EPO pretreatment did not have any effect on these cytokines in C6 cells that were increased by LPS/TNF mixture regardless of the time point.
Effect of EPO on cytotoxicity induced by staurosporine, LPS, and 6-OHDA To determine whether EPO has a cell protective effect against various cytotoxic drugs in C6 cells, LDH assay was performed in C6 cells. As shown in Fig. 4, staurosporine and 6-OHDA induced C6 cell death, but LPS did not. In addition, EPO showed the cell protective effect only in staurosporine-induced cell death, but not in 6-OHDA induced cell death. Thus, we used staurosporine as the C6 cell cytotoxicity model for investigating further signaling mechanisms.
Time course expressions of pAkt, pERK, pP38, pJNK that are induced by EPO treatment To explore the relationship between the cell protective effects of EPO and the PI3K or MAPK pathways, western blot was performed in time course to examine whether EPO increases pAkt and MAPK expression in C6 cells. As shown in Fig. 5A, EPO increased pAkt expression only at 30 minutes after EPO treatment and the signal was not changed at other time points. The pERK and pP38 expression were gradually increased until 2 hours after EPO treatment in C6 cells. However, pJNK was not altered by EPO treatment in C6 cells.
Role of Akt-MAPK pathway on cell protective effect of EPO in staurosporine cytotoxicity We examined whether EPO had anti-inflammatory effects induced by LPS/TNF mixture on C6 cells. Based on the previous study28 that EPO has cell protective effect via various signaling, we investigated the effect of PI3K or MAPK inhibitors on cell protection of EPO in staurosporine-induced cytotoxicity model. As shown in Fig. 5B, the inhibitors PI3K, pP38, and pERK abolished the effects of EPO on cytotoxicity of staurosporine.
Discussion In the present study, we found that EPO has cell protective effects in the C6 cell via the PI3K/Akt and MAPK pathways and not via anti-inflammatory pathways. Our results showed that EPO did not reduce nitrite production, iNOS, TNF-alpha, and IL-1beta mRNA level that were increased by LPS. However, EPO has a cell protective effect on staurosporine-induced apoptosis in LDH assay. In addition, the cell protective effect of EPO was inhibited by PI3K/Akt and MAPK inhibitors, which suggests that these signal molecules have a role in the
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Figure 2 Effect of erythropoietin (EPO) on nitrite production induced by the LPS/TNF-alpha mixture in C6 cells. Various EPO concentrations (0.0005–5 units) were (A) pretreated 24 hours prior to the LPS/TNF-alpha mixture (50 mg/5 mg/well) in C6 cells or (B) co-treated with the LPS/TNF-alpha mixture. The nitrite assay was performed 24 hours after the LPS/TNF-alpha mixture treatment. The nitrite concentration was increased by the LPS/TNF-alpha mixture in C6 cells, and EPO pretreatment or cotreatment did not affect to the nitrite concentrations regardless of EPO concentration. Controls were treated with the vehicle (1% DMSO) and ‘EPO’ was treated with the concentration of 5 units/well. The data are mean¡standard mean error. *** P , 0.001, when compared with the controls.
cell protective effect of EPO against staurosporineinduced apoptosis. Erythropoietin has been widely studied to have a neuroprotective effect in multiple models of nervous system injury, including focal and global ischemia,3 experimental autoimmune encephalomyelitis,1 kainic acid-induced seizure, experimental traumatic brain injury,1 neurotoxin-induced experimental Parkinsonism,28 neonatal hypoxic-ischemic brain injury,29 and peripheral nerve injury.30 These protective mechanisms of EPO against several neurodegenerative or acuteneuronal injuries have extensively characterized in vitro and in vivo models and its direct anti-apoptotic effect and anti-inflammatory effect have been explained as major mechanisms. However, analytical research on the cell-type specific studies has not been conducted yet. Regarding anti-inflammatory mechanisms, EPO has been reported to be a cell protective agent which contributes to the decrease of pro-inflammatory cytokine production in the pro-apoptotic status, such as a cerebral ischemia.4,31 For the control of inflammatory response, pro-inflammatory cytokines, as well as nitric oxide production have been regarded to play important roles in the prevention of inflammatory response that is associated with apoptosis.32 In the CNS, iNOS is expressed mainly in activated astrocytes and microglia. High levels of NO in the CNS are implicated in neuronal death during trauma.33 The effects of EPO in the regulation of NO expression and production have been assumed as
a critical part of the cell protective effect in brain ischemia. In fact, EPO inhibited the iNOS expression in the ischemic hippocampus4 and in primary oligodendrocyte cultures that were exposed to inflammatory stimuli.34 Particularly, EPO suppresses the inflammatory mononuclear cell infiltration and the burst of pro-inflammatory cytokines in a stroke model.35 However, the effect of EPO on iNOS expression and NO production may depend on cell types or conditions. In the C6 cells derived from rat astrocyte, the cell protective effect of EPO appear to be not associated with inhibition of NO, since EPO did not suppress the LPS/TNF mixture-induced NO production in a manner similar to that in microglial cells.36 In addition, EPO did not inhibit the mRNA expressions of pro-inflammatory cytokines (iNOS, TNF-alpha, and IL-beta) that were increased by LPS/ TNF mixture in the C6 cells. The present data indicate that EPO does not have inhibitory activity in the production and the release of inflammatory mediators from C6 cells. Thus, EPO can be speculated to have the cell protective effect by direct EPO/ EPOR interaction, at least in the C6 cells derived from rat astrocyte, in line with previous studies.36 Erythropoietin was shown to have the cell protective mechanisms only against the staurosporine mediated cell toxicity. Cytotoxicity of staurosporine in astrocytes has been reported by previous studies and the p53 and MAPK signaling pathways were found to be involved in abnormal cellular activities that are caused by staurosporine.37 Erythropoietin was found to have cell protective effect against
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Figure 4 Effect of erythropoietin (EPO) against cytotoxicity induced by staurosporine, LPS, or 6-OHDA. Erythropoietin (1 unit/well) was pretreated 24 hours prior to staurosporine (0.2 mM), LPS (50/5 mg/well), or 6-OHDA(300 mM) treatment. LDH assay was performed 6 hours after staurosporine, LPS, or 6-OHDA treatment. Erythropoietin has cell protective effect in staurosporine-induced cytotoxicity, but not in 6OHDA. LPS did not induce cytotoxicity in C6 cells. The data are mean¡standard mean error. * P , 0.05, *** P , 0.001, when compared to the controls.
Figure 3 Effect of erythropoietin (EPO) on iNOS, TNF-alpha, and IL-1beta mRNA expression increased by LPS/TNF-a mixture. The vehicle (DMSO) or EPO (5 units/well) was treated 24 hours prior to LPS/TNF-alpha mixture. After LPS/ TNF-alpha mixture treatment, the real-time RT-PCR was performed to observe the time course (0, 30 minutes, 1 hour, 6 hours, 24 hours) (A) iNOS, (B) TNF-alpha, (C) IL-1beta mRNA expression in C6 cells. iNOS, TNF-alpha, and IL-1beta mRNA expression showed a peak value after LPS/TNF-alpha mixture treatment in C6 cells, and the EPO pretreatment did not affect each cytokine expression. The data are mean¡ standard mean error. *** P , 0.001, ### P , 0.001, when compared with the controls at 0 minute.
neurotoxicity of ketamine by activating the PI3K/Akt pathway.38 When other cytotoxic materials such as 6-OHDA and LPS were introduced, the cell effect of EPO was not observed. 6-OHDA is easily oxidized to reactive oxygen species and induces neuronal death by increasing free radicals.39 LPS is bacterial endotoxin that induces host inflammatory response, and LPS was found to stimulate the cell death by IFNgamma stimulus.40 Since cytotoxic mechanism of 6OHDA and LPS does not involve the p53 and
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MAPK signaling pathways that are mediated by it, EPO seemed to be ineffective in cell protection against 6-OHDA and LPS. A few of the signaling pathways induced by EPO for cell protection are similar to those of erythropoiesis. EPO promotes cell survival by participating in many signaling pathways. The tissue-protective molecular signaling pathways (PI3K/Akt, and MAPK) that are triggered by EPO seem to be important.41 A previous study has found that the PI3K/pAkt signaling pathway is involved in EPO-mediated cell protection against ketamine.1 We examined whether the PI3K/pAkt and MAPK pathways are involved in the cell protection mechanism of EPO. The two signaling pathway may complement each other. In particular, Akt showed a transient phosphorylation 30 minutes after EPO treatment, suggesting that its activation may contribute to continuous activation of MAPK (pERK and pp38) for 2 hours although we did not observe whether this activation was persistent. In addition, the inhibitors of PI3K/Akt and MAPK (pERK and pp38) abolished the cell protective effect of EPO in C6 cells. These results are supported by previous studies that state that the PI3K/Akt and MAPK pathways inhibit the caspase activation for anti-apoptotic effect.31 Taken together, it is speculated that direct EPO/EPOR binding may contribute
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Figure 5 Effect of erythropoietin (EPO) on pAkt, pERK1/2, pP38, pJNK expression and the role of the PI3K/Akt and MAPK signaling pathways on staurosporine-induced cytotoxicity. Western blot (pAkt, pERK1/2, pP38, pJNK) was performed 30 minutes, 1 hour, and 2 hours after EPO (1 unit/well) treatment in (A) C6 cells. The pAkt expression was increased only at 30 minutes after EPO treatment while pERK1/2 and pP38 expression was increased gradually until 2 hours after EPO treatment. (B) LY294002 (50 mM) and wortmannin (1 mM) as PI3K/pAkt inhibitors, SB203580 (10 mM, pP38 inhibitor), or PD98059 (50 mM, pERK inhibitor) was pretreated 1 hour prior to the EPO treatment. Erythropoietin was treated 24 hours before staurosporineinduced cytotoxicity(0.2 mM). LDH assay was performed 6 hours after staurosporine treatment. The data are mean¡standard mean error. ** P , 0.05, *** P , 0.001 when compared to the controls.
to the cell protection via survival signals such as PI3K/ Akt and MAPK in C6 cells although further study for the exact signal pathway needs to be performed. In the CNS system, both EPO and EPOR were expressed in neurons and glial cells of human or rodent brains.24 In particular, astrocytes and neurons express EPO and EPOR,24 whereas microglia does not produce EPO and is only capable of constitutive EPOR mRNA expression42 and protein synthesis.3 Among neurons, astrocytes, and microglia, astrocytes are well known to be the main cellular source of local EPO production,24,25,43 which may act in paracrine or autocrine manner to provide a neuroprotective effect. For this reason, astrocytes can be considered as important targets for neuroprotection in the brain. For example, it has been demonstrated that EPO promotes the maturation and differentiation of oligodendrocytes and the proliferation of astrocytes in vitro.44 In addition, endogenous EPO/EPOR signaling promotes cell survival in the embryonic brain and contributes to neural cell proliferation in adult brain regions for neurogenesis.45 Furthermore,
when endogenous EPO production was blocked by EPO siRNA in astrocyte, the oligodendrocyte survival rate was decreased against hypoxic stress.46 In the present study, we also examined that EPO has the cell protective effects on C6 cells against neurotoxicity (staurosporine). These results suggest that the EPO/ EPOR system may serve as an endogenous system to protect brain cells from damage caused by neurotoxicity. Thus, it is speculated that the protected astrocytes may play an important role in neuronal survival via indirect support, such as the secretion of neuroprotective factors including EPO, although we did not confirm these results in primary astrocyte culture. In conclusion, cell protective function of EPO was only effective in staurosporine mediated cell toxicity, which is mediated by the p53 and MAPK pathways. Erythropoietin seems to have the cell protective effect against cell cytotoxicity by activating the PI3K/Akt and MAPK pathways in C6 cells, and not via antiinflammatory effect. To determine whether these possible mechanisms of the role and sustainment
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time of the PI3K/Akt and MAPK pathways in the cell protective effect of EPO can be actually reconstructed in primary astrocyte culture, further study will be required.
Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A6A3A03040071) and by a grant (10171KFDA336) from Korea Food and Drug Administration in 2010. The views presented in this article do not necessarily reflect those of the KFDA.
References 1 Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle DC, Cerami C, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Nat Acad Sci USA. 2000;97:10526–31. 2 Morishita E, Masuda S, Nagao M, Yasuda Y, Sasaki R. Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience. 1997;76: 105–16. 3 Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, et al. A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab. 1999;19:643–51. 4 Calapai G, Marciano MC, Corica F, Allegra A, Parisi A, Frisina N, et al. Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation. Eur J Pharmacol. 2000;401:349–56. 5 Grasso G, Sfacteria A, Meli F, Fodale V, Buemi M, Iacopino DG. Neuroprotection by erythropoietin administration after experimental traumatic brain injury. Brain Res. 2007;1182: 99–105. 6 Chong ZZ, Li F, Maiese K. Erythropoietin requires NFkappaB and its nuclear translocation to prevent early and late apoptotic neuronal injury during beta-amyloid toxicity. Curr Neurovasc Res. 2005;2:387–99. 7 Xue YQ, Zhao LR, Guo YR, Duan WM. Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson’s disease. Neuroscience. 2007;146:1245–58. 8 Ehrenreich H, Weissenborn K, Prange H, Schneider D, Weimar C, Wartenberg K, et al. Recombinant human erythropoietin in the treatment of acute ischemic stroke. Stroke. 2009;40: e647–56. 9 Nirula R, Diaz-Arrastia R, Brasel K, Weigelt JA, Waxman K. Safety and efficacy of erythropoietin in traumatic brain injury patients: a pilot randomized trial. Crit Care Res Pract. 2010; 2010:5–9. 10 Tseng MY, Hutchinson PJ, Richards HK, Czosnyka M, Pickard JD, Erber WN, et al. Acute systemic erythropoietin therapy to reduce delayed ischemic deficits following aneurysmal subarachnoid hemorrhage: a Phase II randomized, double-blind, placebo-controlled trial. J Neurosurg. 2009; 111:171–80. 11 Ruscher K, Freyer D, Karsch M, Isaev N, Megow D, Sawitzki B, et al. Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model. J Neurosci. 2002;22:10291–301. 12 Siren AL, Knerlich F, Poser W, Gleiter CH, Bruck W, Ehrenreich H. Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain. Acta Neuropathol. 2001; 101:271–6. 13 Bartesaghi S, Marinovich M, Corsini E, Galli CL, Viviani E. Erythropoietin: a novel neuroprotective cytokine. Neurotoxicology. 2005;26:923–8. 14 Genc S, Koroglu TF, Genc K. Erythropoietin and the nervous system. Brain Res. 2004;1000:19–31. 15 Zhang F, Signore AP, Zhou Z, Wang S, Cao G, Chen J. Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: potential signaling mechanisms. J Neurosci Res. 2006;83:1241–51.
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16 Attwell D, Buchan AM, Charpak S, Lauritzen M, Macvicar BA, Newman EA. Glial and neuronal control of brain blood flow. Nature. 2010;468:232–43. 17 Carnevale D, De Simone R, Minghetti L. Microglia-neuron interaction in inflammatory and degenerative diseases: role of cholinergic and noradrenergic systems. CNS Neurol Disord Drug Targets. 2007;6:388–97. 18 Guo S, Lo EH. Dysfunctional cell-cell signaling in the neurovascular unit as a paradigm for central nervous system disease. Stroke. 2009;40:S4–7. 19 Maragakis NJ, Rothstein JD. Mechanisms of disease: astrocytes in neurodegenerative disease. Nat Clin Pract Neurol. 2006;2:679–89. 20 Takuma K, Baba A, Matsuda T. Astrocyte apoptosis: implications for neuroprotection. Prog Neurobiol. 2004; 72:111–27. 21 Benda P, Lightbody J, Sato G, Levine L, Sweet W. Differentiated rat glial cell strain in tissue culture. Science. 1968;161(3839):370–1. 22 Haghighat N, Oblinger MM, McCandless DW. Cytoprotective effect of estrogen on ammonium chloride-treated C6-glioma cells. Neurochem Res. 2004;29(7):1359–64. 23 Marti HH, Wenger RH, Rivas LA, Straumann U, Digicaylioglu M, Henn V, et al. Erythropoietin gene expression in human, monkey and murine brain. Eur J Neurosci. 1996;8:666–76. 24 Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R. A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J Biol Chem. 1994;269:19488–93. 25 Feinstein DL, Galea E, Roberts S, Berquist H, Wang H, Reis DJ. Induction of nitric oxide synthase in rat C6 glioma cells. J Neurochem. 1994;62:315–21. 26 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–9. 27 Mitchell DB, Acosta D. Evaluation of the cytotoxicity of tricyclic antidepressants in primary cultures of rat hepatocytes. J Toxic Environ Health. 1981;7:83–92. 28 Genc K. Erythropoietin and Parkinson’s disease: suggested mechanisms and therapeutic implications. Med Hypotheses. 2008;70:211–2. 29 Kumral A, Ozer E, Yilmaz O, Akhisaroglu M, Gokmen N, Duman N, et al. Neuroprotective effect of erythropoietin on hypoxic-ischemic brain injury in neonatal rats. Biol Neonate. 2003;83:224–8. 30 Iwasaki Y, Ikeda K, Ichikawa Y, Igarashi O, Iwamoto K, Kinoshita M. Protective effect of interleukin-3 and erythropoietin on motor neuron death after neonatal axotomy. Neurol Res. 2002;24:643–6. 31 Brines M, Cerami A. Emerging biological roles for erythropoietin in the nervous system. Nat Rev Neurosci. 2005;6: 484–94. 32 Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature. 2001;412:641–7. 33 Mitrovic B, Ignarro LJ, Montestruque S, Smoll A, Merrill JE. Nitric oxide as a potential pathological mechanism in demyelination: its differential effects on primary glial cells in vitro. Neuroscience. 1994;61:575–85. 34 Genc K, Genc S, Baskin H, Semin I. Erythropoietin decreases cytotoxicity and nitric oxide formation induced by inflammatory stimuli in rat oligodendrocytes. Physiol Res. 2006;55:33– 8. 35 Villa P, Bigini P, Mennini T, Agnello D, Laragione T, Cagnotto A, et al. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med. 2003;198:971–5. 36 Vairano M, Dello Russo C, Pozzoli G, Battaglia A, Scambia G, Tringali G, et al. Erythropoietin exerts anti-apoptotic effects on rat microglial cells in vitro. Eur J Neurosci. 2002;16:584–92. 37 Simenc J, Lipnik-Stangelj M. Staurosporine induces different cell death forms in cultured rat astrocytes. Radiol Oncol. 2012;46(4):312–20. 38 Shang Y, Wu Y, Yao S, Wang X, Feng D, Yang W. Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: involvement of PI3K/Akt and GSK-3 beta pathway. Apoptosis. 2007;12(12):2187–95. 39 Lotharius J, Dugan LL, O’Malley KL. Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons. J Neurosci. 1999;19(4):1284–93.
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40 Dijkmans R, Van Damme J, Cornette F, Heremans H, Billiau A. Bacterial lipopolysaccharide potentiates gamma interferoninduced cytotoxicity for normal mouse and rat fibroblasts. Infect Immun. 1990;58(1):32–6. 41 Fisher JW. Erythropoietin: physiology and pharmacology update. Exp Biol Med (Maywood). 2003;228:1–14. 42 Nagai A, Nakagawa E, Choi HB, Hatori K, Kobayashi S, Kim SU. Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture. J Neuropathol Exp Neurol. 2001;60:386–92. 43 Bernaudin M, Bellail A, Marti HH, Yvon A, Vivien D, Duchatelle I, et al. Neurons and astrocytes express EPO
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mRNA: oxygen-sensing mechanisms that involve the redoxstate of the brain. Glia. 2000;30:271–8. 44 Sugawa M, Sakurai Y, Ishikawa-Ieda Y, Suzuki H, Asou H. Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation. Neurosci Res. 2002;44:391–403. 45 Chen ZY, Asavaritikrai P, Prchal JT, Noguchi CT. Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation. J Biol Chem. 2007;282:25875–83. 46 Kato S, Aoyama M, Kakita H, Hida H, Kato I, Ito T, et al. Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury. J Neurol Res. 2011;89:1566–74.
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Erythropoietin exerts cell protective effect by activating PI3K/Akt and MAPK pathways in C6 Cells Min-Soo Kwon1, Mi-Hee Kim2, Seon-Hee Kim2, Ki-Dae Park2, Si-Hyung Yoo2, Il-Ung Oh2, Suenie Pak2, Young-Jun Seo3 1
Department of Pharmacology, School of Medicine, CHA University, Yatap-dong, Bundang-gu, Seongnam, Gyeonggi-do, Republic of Korea, 2Advanced Therapy Products Research Division Pharmaceutical and Medical Device Research Department, NIFDS Korea Food and Drug Administration, Gangoe-myeon, Cheongwon-gun, Chungcheongbuk-do, Republic of Korea, 3Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Even though erythropoietin (EPO) is a neurotropic cytokine that is recognized widely for its role in the development, maintenance, protection, and repair of the nervous system, there are few reports concerning EPO-mediated influences on the glial cells in the central nervous system. In this study, we investigated antiinflammatory and anti-apoptotic effects of EPO on C6 glioma cells (C6 cells). Erythropoietin did not attenuate inflammatory response, such as nitrite production, iNOS gene expression, and pro-inflammatory cytokines when LPS/TNF-alpha mixture was treated. However, EPO increased C6 cell viability by exerting cell protective effect against staurosporine stimulation. Erythropoietin increased the transient Akt expression at 30 minutes and induced the gradual elevation of ERK1/2 and p38 expression as time progressed. The cell protective effect of EPO was also significantly attenuated with pretreatment of specific PI3K, pERK1/2, or pP38 inhibitor. In summary, these results suggest that EPO may exert its cell protective functions via the direct cell protective activity rather than via its anti-inflammatory effect. Moreover, the PI3K/Akt and mitogen activated protein kinase (MAPK) pathways may be responsible for cell survival against cytotoxicity. Keywords: Erythropoietin, C6 cells, Cell protection, Anti-inflammation, Signal pathway
Introduction Erythropoietin (EPO) has been originally characterized as a major hematopoietic growth factor, but recently it has been proposed to be one of the most efficient neuroprotective agents as well. Several in vitro/vivo studies have provided evidence for the critical role of EPO in the control of neuronal function and have suggested a potential neuroprotective value in CNS disorders with various pathophysiological origins, such as neurotoxic and excitotoxic stress,1,2 cerebral ischemia, hypoxia,3,4 trauma,5 and inflammatory and neurodegenerative diseases.6,7 Based on these experimental reports, EPO has undergone evaluations in clinical trials for patients with various neurological diseases, such as stroke, traumatic brain injury, and subarachnoid hemorrhage.8–10
Correspondence to: Young-jun Seo, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Email: [email protected]
ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132813Y.0000000284
Erythropoietin binding causes the EPO receptor (EPOR) to form a homodimeric or EPOR–beta common receptor (BCR) heterotrimeric structure that triggers complex intracellular signal cascades, such as JAK2/STAT5 signaling, phosphatidylinositol 3-kinase (PI3K) signaling, protein kinase C, mitogen activated protein kinase (MAPK), and nuclear factor (NF)-kB pathways.1,6,11,12 The EPOR stimulation in the target cells leads to the proliferation and differentiation of stem cells, the inhibition of apoptosis, and anti-inflammatory and anti-oxidative effects. In the nervous system, mechanisms including direct neuroprotection, anti-inflammation, anti-apoptotic, antioxidative effects, stimulation of neurogenesis, and modulation of neurotransmission have been suggested to be crucial bases underlying the beneficial effects of EPO.13,14,15 Astrocytes, the most prevalent glial cell types in the brain, provide metabolic/trophic support to neurons and modulate synapse. Therefore, impairment in these astrocyte functions can critically influence neuronal
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survival. Recent studies have demonstrated that apoptosis of astrocytes may contribute to pathogenesis of many acute or chronic neurodegenerative diseases, such as cerebral ischemia, Alzheimer’s disease, and Parkinson’s disease.16–19 In addition, astrocytes have high concentration of antioxidants, such as low mole>cular weight antioxidants (e.g., ascorbic acid and glutathione peroxidase) and antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), which protect the surrounding cells containing neurons from oxidant stress-induced cell death.20 As a model mimicking astrocyte properties, C6 glioma cell line has been used frequently.21 Additionally, later stages of C6 glioma cells exhibit several characteristics that resemble astrocytes. Therefore, C6 glioma cells have been used as an alternative cell model for astrocytes owing to its convenience for rapid screening.22 Even though other brain cells, such as neurons, oligodendrocytes, microglia, and endothelial cells can synthesize EPO in response to hypoxia-ischemia or other stressors, astrocytes are the main cellular source of local EPO.23,24 The local production of endogenous EPO may act in paracrine and autocrine manners to provide a neuroprotective effect. Since anti-oxidative effects are involved in the neuroprotective process of EPO in the endothelial cells, this raises the possibility that EPO may also play a role in cellular defenses against oxidative stress and subsequent apoptosis in astrocytes. In addition, the neuroprotective effects of EPO have been described in various models of neurotoxicity, but there is limited evidence regarding EPO-mediated influences on glial cells. Thus, we investigated the anti-apoptotic and anti-inflammatory effects of EPO against various toxic and inflammatory stimulations in cultured C6 glioma cells (C6 cells) mimicking astrocytes. We also examined the possible mechanisms of these effects.
Materials and Methods Cell culture and drug treatment Rat C6 glioma cells were obtained from the Korea Cell Line Bank and maintained in Dulbecco’s modified Eagle’s medium/F12 (Gibco, USA) containing 10% heat-inactivated fetal bovine serum (Gibco, USA), 2.2 g/l sodium bicarbonate, 0.6% (w/v) Dglucose, and 50 mg/ml penicillin and streptomycin. Cells were plated on 25-cm2 culture flasks (Corning, USA). The cultures were incubated at 37uC in 5% CO2 for 3 days. The cells were incubated with a serum-free culture medium for 24 hour prior to the incubation with drugs. Various EPO doses (0.0005–5 units/well) were co-treated with the LPS/TNF mixture at the same time or were pretreated 24 hours prior with the LPS/TNF mixture (50/5 mg/well) in 96 well plates.
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Each experiment was repeated for at least three different cultures.
Immunofluorescence C6 cells were plated onto cover slips that were precoated with collagen (Sigma-Aldrich, St Louis, MO, USA) in DMEM containing 10% FBS and P/S and were incubated at 37uC in humidified 5% CO2. After incubation, the cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 minutes at room temperature. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% and blocked with 10% serum for 60 minutes at 24uC. Samples were incubated with a primary antibody (1:100) for 16 hours at 4uC. Fixed cells were preincubated in 10% normal goat serum for 30 minutes to block nonspecific staining. For the staining of EPOR, the cells were incubated with rabbit antibodies against EPOR (abcam #ab61162, USA) for a minimum of 1 hour at room temperature. After washing with PBS, they were stained with fluorescein isothiocyanate-labeled antibodies against mouse IgG or Texas red-labeled antibodies against rabbit IgG and coverslipped with VECTASHIELD Mounting Media with DAPI. All specimens were observed under a confocal laser-scanning microscope (Olympus LX70 fluorescent microscope equipped with a HXP 120 C Hg lamp and DAPI and RFP filter-set).
Assay for NO synthesis NO synthesis was determined by an assay that detects nitrite, a stable reaction product of NO with molecular oxygen, in culture supernatant. Briefly, 100 ml of culture supernatant was allowed to react with 100 ml of Griess reagent25 and incubated at room temperature for 15 minutes. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture media served as blanks in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay.
Isolation of total RNA Total cellular RNA was extracted from C6 glioma cells using a rapid guanidine thiocyanate–water-saturated phenol/chloroform extraction procedure and subsequent precipitation with acidic sodium acetate.26 Total cellular RNA in the aqueous phase was precipitated with ice-cold isopropyl alcohol. Isolated RNA samples were subjected to spectrophotometric analysis at 260 and 280 nm. The separated organic layer was extracted twice with an equal volume of sterilized water (Millipore, Billerica, MA, US). Adding two volumes of absolute ethanol to the water-extracted organic phase precipitated the proteins. The RNA pellets were washed twice with cold absolute ethanol and dried. The dried pellets were
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dissolved in a denaturing buffer (6 M guanidinium chloride, 20 mM Tris–HCl [pH 8.0], and 1 mM EDTA). The RNA samples were dialyzed against a renaturing buffer (20 mM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 0.4 mM phenylmethylsulfonyl fluoride, and 20% glycerol) at 4uC. The concentration of RNA was determined with the Coomassie Blue protein assay reagent (Pierce Chemical Co., Rockford, IL, USA) using bovine serum albumin (BSA) as the standard.
Real-time PCR analysis The expression of TNF-alpha, IL-1beta, iNOS and GAPDH mRNA was evaluated by real-time PCR using QuantiTectTM SYBRH Green PCR Kit (Qiagen, Germany). All PCRs were performed in a total volume of 20 ml using the QuantiTectTM SYBR Green PCR Kit (Qiagen, Germany). Each reaction contained 1.5 ml of cDNA, 6.5 ml RNasefree water, 1 ml each of sense and antisense primers (20 mM), and 10 ml of 26 SYBR Green PCR Master Mix (containing QuantiTect SYBR Green PCR buffer, dNTPs, SYBR Green I dye, ROX dye, and HotStarTaq DNA polymerase). After an initial denaturation step at 95uC for 30 seconds, temperature cycling with a total of 40 cycles was initiated. Each cycle consisted of a denaturation phase at 95uC for 30 seconds, an annealing phase at 60uC for 30 seconds, and an elongation phase at 72uC for 30 seconds. Amplification was followed by a melting curve analysis to verify the correctness of the amplification. A negative control with water instead of cDNA was run within every PCR to assess the specificity of the reaction. To verify the accuracy of the amplification, PCR products were further analyzed on ethidium bromide-stained 2% agarose gel. For data analysis, Rotor-Gene 6000 Series Software 1.7 (Build 87) was used. Results are given as a ratio of the amount of TNF-alpha, IL-1beta, iNOS mRNA to that of GAPDH mRNA. The following primers were used: TNF-alpha (NM 012675) sense: AAC TCG AGT GAC AAG CCC GTA G, antisense: GTA CCA CCA GTT GGT TGT CTT TGA; IL-1beta (NM 031512) sense: GCT GTG GCA GCT ACC TAT GTC TTG, antisense: AGG TCG TCA TCA TCC CAC GAG; iNOS (NM 012611) sense: CTG TGT GCC TGG AGG TTC TAG ATG, antisense: AAG TAG GTG AGG GCT TGC CTG A; and GAPDH (NM 017008) sense: GGC ACA GTC AAG GCT GAG AAT G, antisense: ATG GTG GTG AAG ACG CCA GTA. The entire process to RTPCR was conducted three times independently. Each real-time PCR result (independently three times) was quantified with GraphPad Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA, USA) for statistical analysis.
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Western blot analysis The cells were washed with ice-cold Tris-buffered saline (TBS; 20 mM Trizma base and 137 mM NaCl, pH 7.5) and lysed in 16SDS sample loading buffer (62.5 mM Trizma base, 2% w/v SDS, 10% glycerol); after sonication and centrifugation at 15 0006g for 5 minutes, the supernatant was used for western blot assay. The protein concentrations of the samples were determined with a detergent-compatible protein assay reagent (Bio-Rad, Hercules, CA, USA), using BSA as standard. Samples were boiled for 3 minutes with 0.1 volume of 10% beta-mercaptoethanol and 0.5% bromophenol blue mix. Of the total cellular protein, 50 mg was resolved by electrophoresis on 8% polyacrylamide gels, electro-transferred to a PVDF membrane (Amersham Pharmacia, Buckinghamshire, UK) and blocked with TBST (10 mM Trizma base (pH 7.4), 150 mM NaCl, and 1% Tween 20) with 5% skim milk. After incubation with antiserum against pAkt, pERK, pP38, pJNK, Akt, and betaactin (#9271, #9102, #9211, #9251, #4691, #4967; Cell Signal Technology, CA, USA) for 2 hours at room temperature, the membranes were washed with TBST three times and then incubated with horseradish peroxidase conjugated anti-rabbit IgG for 1 hour. The membranes were visualized by using ECL-plus (Amersham Pharmacia) after washing with TBST buffer.
LDH assay LDH leakage from damaged cells was measured using a Roche LDA assay kit that quantitatively measures LDH, a stable cytosolic enzyme that is released upon cell lysis.27 The cells were treated with EPO 24 hours after seeding (1 unit/well) and incubated for another 24 hours Then, 50 ml of cell culture medium was collected from each well and plated on a new microtiter plate. Next, 50 ml of staurosporine (0.2 mM) was added to the wells, and the plates were incubated for 30 minutes at room temperature. The optical density at 490 nm was measured with a standard microplate reader. Each experiment was performed in triplicate. The LDH leakage (%) relative to the control wells containing cell culture medium without EPO and staurosporine, was calculated as [test][control] 100, where [test] was the absorbance of the test sample and [control] was the absorbance of the control sample
Drugs SB203580, PD98059, wortmannin and LY294002 were purchased from Cellsignal Technology (#5633, #9900, #9951, 9901; USA). Staurosporine was purchased from Roche Applied Science, IN, USA. SB203580, PD98059, wortmannin, LY294002, and staurosporine were dissolved in 1% DMSO solution that was diluted with the DMEM/F12 media.
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Figure 1 The expression of erythropoietin receptor (EPOR) in C6 cells. Immunofluorescence staining of C6 cells with an anti-EPOR antibody was performed. The EPOR was well expressed in the C6 cells derived from rat astrocytes.
LY294002 (50 mM) and wortmannin (1 mM) as PI3K/ pAkt inhibitors, SB203580 (10 mM, pP38 inhibitor), or PD98059 (50 mM, pERK inhibitor) was pretreated 1 hour prior to the EPO treatment. Drug doses of SB203580, PD98059, wortmannin, and LY294002, which were determined as critical concentrations for inhibition, were chosen based on the product reference data and previously published research. The controls were pretreated with 20% DMSO solution that was diluted with the DMEM/F12 media.
Statistical Analysis Data were presented as mean¡SEM. The statistical significance of differences between groups was assessed with one-way ANOVA with Bonferroni’s post hoc test or t-tests (and nonparametic tests) using GraphPad Prism version 5.00 for Mac OS X, (GraphPad Software, San Diego California USA, www.graphpad.com); P , 0.05 was considered significant.
Results Erythropoietin receptor expression in C6 cells To evaluate EPOR expression in C6 cells, we performed the confocal study. As shown in Fig. 1, EPOR expression was confirmed in C6 cells.
Effect of EPO on nitrite production increased by LPS/TNF mixture in C6 cells To observe whether EPO has an inhibitory effect on nitrite production in C6 cells with LPS/TNF mixture, we pretreated or co-treated EPO in C6 cells with LPS/ TNF, and then nitrite assay was performed. As the effect of EPO may depend on the concentration and treatment time in the C6 cell with the LPS/TNF mixture, we pretreated or co-treated the EPO in various doses. As shown in Fig. 2, pre or co-treated EPO did not have any effect on the increase in nitrite concentration in C6 cells with the LPS/TNF mixture regardless of its concentration (0.0005–5 units/well).
Effect of EPO on iNOS, TNF-alpha, and IL-beta mRNA expression induced by LPS/TNF mixture in C6 cells To observe the anti-inflammatory effect of EPO on mRNA levels of pro-inflammatory cytokines, we pretreated a vehicle (20% DMSO in media) or EPO in C6 cells 24 hours prior to the LPS/TNF treatment. The iNOS, TNF-alpha, and IL-1beta mRNA level
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were examined over several time points (30 minutes, 1 hour, 6 hours, 24 hours). As shown in Fig. 3, iNOS, TNF-alpha, and IL-1beta showed a peak level 6 hours after the LPS/TNF mixture treatment. However, EPO pretreatment did not have any effect on these cytokines in C6 cells that were increased by LPS/TNF mixture regardless of the time point.
Effect of EPO on cytotoxicity induced by staurosporine, LPS, and 6-OHDA To determine whether EPO has a cell protective effect against various cytotoxic drugs in C6 cells, LDH assay was performed in C6 cells. As shown in Fig. 4, staurosporine and 6-OHDA induced C6 cell death, but LPS did not. In addition, EPO showed the cell protective effect only in staurosporine-induced cell death, but not in 6-OHDA induced cell death. Thus, we used staurosporine as the C6 cell cytotoxicity model for investigating further signaling mechanisms.
Time course expressions of pAkt, pERK, pP38, pJNK that are induced by EPO treatment To explore the relationship between the cell protective effects of EPO and the PI3K or MAPK pathways, western blot was performed in time course to examine whether EPO increases pAkt and MAPK expression in C6 cells. As shown in Fig. 5A, EPO increased pAkt expression only at 30 minutes after EPO treatment and the signal was not changed at other time points. The pERK and pP38 expression were gradually increased until 2 hours after EPO treatment in C6 cells. However, pJNK was not altered by EPO treatment in C6 cells.
Role of Akt-MAPK pathway on cell protective effect of EPO in staurosporine cytotoxicity We examined whether EPO had anti-inflammatory effects induced by LPS/TNF mixture on C6 cells. Based on the previous study28 that EPO has cell protective effect via various signaling, we investigated the effect of PI3K or MAPK inhibitors on cell protection of EPO in staurosporine-induced cytotoxicity model. As shown in Fig. 5B, the inhibitors PI3K, pP38, and pERK abolished the effects of EPO on cytotoxicity of staurosporine.
Discussion In the present study, we found that EPO has cell protective effects in the C6 cell via the PI3K/Akt and MAPK pathways and not via anti-inflammatory pathways. Our results showed that EPO did not reduce nitrite production, iNOS, TNF-alpha, and IL-1beta mRNA level that were increased by LPS. However, EPO has a cell protective effect on staurosporine-induced apoptosis in LDH assay. In addition, the cell protective effect of EPO was inhibited by PI3K/Akt and MAPK inhibitors, which suggests that these signal molecules have a role in the
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Figure 2 Effect of erythropoietin (EPO) on nitrite production induced by the LPS/TNF-alpha mixture in C6 cells. Various EPO concentrations (0.0005–5 units) were (A) pretreated 24 hours prior to the LPS/TNF-alpha mixture (50 mg/5 mg/well) in C6 cells or (B) co-treated with the LPS/TNF-alpha mixture. The nitrite assay was performed 24 hours after the LPS/TNF-alpha mixture treatment. The nitrite concentration was increased by the LPS/TNF-alpha mixture in C6 cells, and EPO pretreatment or cotreatment did not affect to the nitrite concentrations regardless of EPO concentration. Controls were treated with the vehicle (1% DMSO) and ‘EPO’ was treated with the concentration of 5 units/well. The data are mean¡standard mean error. *** P , 0.001, when compared with the controls.
cell protective effect of EPO against staurosporineinduced apoptosis. Erythropoietin has been widely studied to have a neuroprotective effect in multiple models of nervous system injury, including focal and global ischemia,3 experimental autoimmune encephalomyelitis,1 kainic acid-induced seizure, experimental traumatic brain injury,1 neurotoxin-induced experimental Parkinsonism,28 neonatal hypoxic-ischemic brain injury,29 and peripheral nerve injury.30 These protective mechanisms of EPO against several neurodegenerative or acuteneuronal injuries have extensively characterized in vitro and in vivo models and its direct anti-apoptotic effect and anti-inflammatory effect have been explained as major mechanisms. However, analytical research on the cell-type specific studies has not been conducted yet. Regarding anti-inflammatory mechanisms, EPO has been reported to be a cell protective agent which contributes to the decrease of pro-inflammatory cytokine production in the pro-apoptotic status, such as a cerebral ischemia.4,31 For the control of inflammatory response, pro-inflammatory cytokines, as well as nitric oxide production have been regarded to play important roles in the prevention of inflammatory response that is associated with apoptosis.32 In the CNS, iNOS is expressed mainly in activated astrocytes and microglia. High levels of NO in the CNS are implicated in neuronal death during trauma.33 The effects of EPO in the regulation of NO expression and production have been assumed as
a critical part of the cell protective effect in brain ischemia. In fact, EPO inhibited the iNOS expression in the ischemic hippocampus4 and in primary oligodendrocyte cultures that were exposed to inflammatory stimuli.34 Particularly, EPO suppresses the inflammatory mononuclear cell infiltration and the burst of pro-inflammatory cytokines in a stroke model.35 However, the effect of EPO on iNOS expression and NO production may depend on cell types or conditions. In the C6 cells derived from rat astrocyte, the cell protective effect of EPO appear to be not associated with inhibition of NO, since EPO did not suppress the LPS/TNF mixture-induced NO production in a manner similar to that in microglial cells.36 In addition, EPO did not inhibit the mRNA expressions of pro-inflammatory cytokines (iNOS, TNF-alpha, and IL-beta) that were increased by LPS/ TNF mixture in the C6 cells. The present data indicate that EPO does not have inhibitory activity in the production and the release of inflammatory mediators from C6 cells. Thus, EPO can be speculated to have the cell protective effect by direct EPO/ EPOR interaction, at least in the C6 cells derived from rat astrocyte, in line with previous studies.36 Erythropoietin was shown to have the cell protective mechanisms only against the staurosporine mediated cell toxicity. Cytotoxicity of staurosporine in astrocytes has been reported by previous studies and the p53 and MAPK signaling pathways were found to be involved in abnormal cellular activities that are caused by staurosporine.37 Erythropoietin was found to have cell protective effect against
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Figure 4 Effect of erythropoietin (EPO) against cytotoxicity induced by staurosporine, LPS, or 6-OHDA. Erythropoietin (1 unit/well) was pretreated 24 hours prior to staurosporine (0.2 mM), LPS (50/5 mg/well), or 6-OHDA(300 mM) treatment. LDH assay was performed 6 hours after staurosporine, LPS, or 6-OHDA treatment. Erythropoietin has cell protective effect in staurosporine-induced cytotoxicity, but not in 6OHDA. LPS did not induce cytotoxicity in C6 cells. The data are mean¡standard mean error. * P , 0.05, *** P , 0.001, when compared to the controls.
Figure 3 Effect of erythropoietin (EPO) on iNOS, TNF-alpha, and IL-1beta mRNA expression increased by LPS/TNF-a mixture. The vehicle (DMSO) or EPO (5 units/well) was treated 24 hours prior to LPS/TNF-alpha mixture. After LPS/ TNF-alpha mixture treatment, the real-time RT-PCR was performed to observe the time course (0, 30 minutes, 1 hour, 6 hours, 24 hours) (A) iNOS, (B) TNF-alpha, (C) IL-1beta mRNA expression in C6 cells. iNOS, TNF-alpha, and IL-1beta mRNA expression showed a peak value after LPS/TNF-alpha mixture treatment in C6 cells, and the EPO pretreatment did not affect each cytokine expression. The data are mean¡ standard mean error. *** P , 0.001, ### P , 0.001, when compared with the controls at 0 minute.
neurotoxicity of ketamine by activating the PI3K/Akt pathway.38 When other cytotoxic materials such as 6-OHDA and LPS were introduced, the cell effect of EPO was not observed. 6-OHDA is easily oxidized to reactive oxygen species and induces neuronal death by increasing free radicals.39 LPS is bacterial endotoxin that induces host inflammatory response, and LPS was found to stimulate the cell death by IFNgamma stimulus.40 Since cytotoxic mechanism of 6OHDA and LPS does not involve the p53 and
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MAPK signaling pathways that are mediated by it, EPO seemed to be ineffective in cell protection against 6-OHDA and LPS. A few of the signaling pathways induced by EPO for cell protection are similar to those of erythropoiesis. EPO promotes cell survival by participating in many signaling pathways. The tissue-protective molecular signaling pathways (PI3K/Akt, and MAPK) that are triggered by EPO seem to be important.41 A previous study has found that the PI3K/pAkt signaling pathway is involved in EPO-mediated cell protection against ketamine.1 We examined whether the PI3K/pAkt and MAPK pathways are involved in the cell protection mechanism of EPO. The two signaling pathway may complement each other. In particular, Akt showed a transient phosphorylation 30 minutes after EPO treatment, suggesting that its activation may contribute to continuous activation of MAPK (pERK and pp38) for 2 hours although we did not observe whether this activation was persistent. In addition, the inhibitors of PI3K/Akt and MAPK (pERK and pp38) abolished the cell protective effect of EPO in C6 cells. These results are supported by previous studies that state that the PI3K/Akt and MAPK pathways inhibit the caspase activation for anti-apoptotic effect.31 Taken together, it is speculated that direct EPO/EPOR binding may contribute
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Figure 5 Effect of erythropoietin (EPO) on pAkt, pERK1/2, pP38, pJNK expression and the role of the PI3K/Akt and MAPK signaling pathways on staurosporine-induced cytotoxicity. Western blot (pAkt, pERK1/2, pP38, pJNK) was performed 30 minutes, 1 hour, and 2 hours after EPO (1 unit/well) treatment in (A) C6 cells. The pAkt expression was increased only at 30 minutes after EPO treatment while pERK1/2 and pP38 expression was increased gradually until 2 hours after EPO treatment. (B) LY294002 (50 mM) and wortmannin (1 mM) as PI3K/pAkt inhibitors, SB203580 (10 mM, pP38 inhibitor), or PD98059 (50 mM, pERK inhibitor) was pretreated 1 hour prior to the EPO treatment. Erythropoietin was treated 24 hours before staurosporineinduced cytotoxicity(0.2 mM). LDH assay was performed 6 hours after staurosporine treatment. The data are mean¡standard mean error. ** P , 0.05, *** P , 0.001 when compared to the controls.
to the cell protection via survival signals such as PI3K/ Akt and MAPK in C6 cells although further study for the exact signal pathway needs to be performed. In the CNS system, both EPO and EPOR were expressed in neurons and glial cells of human or rodent brains.24 In particular, astrocytes and neurons express EPO and EPOR,24 whereas microglia does not produce EPO and is only capable of constitutive EPOR mRNA expression42 and protein synthesis.3 Among neurons, astrocytes, and microglia, astrocytes are well known to be the main cellular source of local EPO production,24,25,43 which may act in paracrine or autocrine manner to provide a neuroprotective effect. For this reason, astrocytes can be considered as important targets for neuroprotection in the brain. For example, it has been demonstrated that EPO promotes the maturation and differentiation of oligodendrocytes and the proliferation of astrocytes in vitro.44 In addition, endogenous EPO/EPOR signaling promotes cell survival in the embryonic brain and contributes to neural cell proliferation in adult brain regions for neurogenesis.45 Furthermore,
when endogenous EPO production was blocked by EPO siRNA in astrocyte, the oligodendrocyte survival rate was decreased against hypoxic stress.46 In the present study, we also examined that EPO has the cell protective effects on C6 cells against neurotoxicity (staurosporine). These results suggest that the EPO/ EPOR system may serve as an endogenous system to protect brain cells from damage caused by neurotoxicity. Thus, it is speculated that the protected astrocytes may play an important role in neuronal survival via indirect support, such as the secretion of neuroprotective factors including EPO, although we did not confirm these results in primary astrocyte culture. In conclusion, cell protective function of EPO was only effective in staurosporine mediated cell toxicity, which is mediated by the p53 and MAPK pathways. Erythropoietin seems to have the cell protective effect against cell cytotoxicity by activating the PI3K/Akt and MAPK pathways in C6 cells, and not via antiinflammatory effect. To determine whether these possible mechanisms of the role and sustainment
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time of the PI3K/Akt and MAPK pathways in the cell protective effect of EPO can be actually reconstructed in primary astrocyte culture, further study will be required.
Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A6A3A03040071) and by a grant (10171KFDA336) from Korea Food and Drug Administration in 2010. The views presented in this article do not necessarily reflect those of the KFDA.
References 1 Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle DC, Cerami C, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Nat Acad Sci USA. 2000;97:10526–31. 2 Morishita E, Masuda S, Nagao M, Yasuda Y, Sasaki R. Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience. 1997;76: 105–16. 3 Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, et al. A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab. 1999;19:643–51. 4 Calapai G, Marciano MC, Corica F, Allegra A, Parisi A, Frisina N, et al. Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation. Eur J Pharmacol. 2000;401:349–56. 5 Grasso G, Sfacteria A, Meli F, Fodale V, Buemi M, Iacopino DG. Neuroprotection by erythropoietin administration after experimental traumatic brain injury. Brain Res. 2007;1182: 99–105. 6 Chong ZZ, Li F, Maiese K. Erythropoietin requires NFkappaB and its nuclear translocation to prevent early and late apoptotic neuronal injury during beta-amyloid toxicity. Curr Neurovasc Res. 2005;2:387–99. 7 Xue YQ, Zhao LR, Guo YR, Duan WM. Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson’s disease. Neuroscience. 2007;146:1245–58. 8 Ehrenreich H, Weissenborn K, Prange H, Schneider D, Weimar C, Wartenberg K, et al. Recombinant human erythropoietin in the treatment of acute ischemic stroke. Stroke. 2009;40: e647–56. 9 Nirula R, Diaz-Arrastia R, Brasel K, Weigelt JA, Waxman K. Safety and efficacy of erythropoietin in traumatic brain injury patients: a pilot randomized trial. Crit Care Res Pract. 2010; 2010:5–9. 10 Tseng MY, Hutchinson PJ, Richards HK, Czosnyka M, Pickard JD, Erber WN, et al. Acute systemic erythropoietin therapy to reduce delayed ischemic deficits following aneurysmal subarachnoid hemorrhage: a Phase II randomized, double-blind, placebo-controlled trial. J Neurosurg. 2009; 111:171–80. 11 Ruscher K, Freyer D, Karsch M, Isaev N, Megow D, Sawitzki B, et al. Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model. J Neurosci. 2002;22:10291–301. 12 Siren AL, Knerlich F, Poser W, Gleiter CH, Bruck W, Ehrenreich H. Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain. Acta Neuropathol. 2001; 101:271–6. 13 Bartesaghi S, Marinovich M, Corsini E, Galli CL, Viviani E. Erythropoietin: a novel neuroprotective cytokine. Neurotoxicology. 2005;26:923–8. 14 Genc S, Koroglu TF, Genc K. Erythropoietin and the nervous system. Brain Res. 2004;1000:19–31. 15 Zhang F, Signore AP, Zhou Z, Wang S, Cao G, Chen J. Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: potential signaling mechanisms. J Neurosci Res. 2006;83:1241–51.
222
Neurological Research
2014
VOL .
36
NO .
3
16 Attwell D, Buchan AM, Charpak S, Lauritzen M, Macvicar BA, Newman EA. Glial and neuronal control of brain blood flow. Nature. 2010;468:232–43. 17 Carnevale D, De Simone R, Minghetti L. Microglia-neuron interaction in inflammatory and degenerative diseases: role of cholinergic and noradrenergic systems. CNS Neurol Disord Drug Targets. 2007;6:388–97. 18 Guo S, Lo EH. Dysfunctional cell-cell signaling in the neurovascular unit as a paradigm for central nervous system disease. Stroke. 2009;40:S4–7. 19 Maragakis NJ, Rothstein JD. Mechanisms of disease: astrocytes in neurodegenerative disease. Nat Clin Pract Neurol. 2006;2:679–89. 20 Takuma K, Baba A, Matsuda T. Astrocyte apoptosis: implications for neuroprotection. Prog Neurobiol. 2004; 72:111–27. 21 Benda P, Lightbody J, Sato G, Levine L, Sweet W. Differentiated rat glial cell strain in tissue culture. Science. 1968;161(3839):370–1. 22 Haghighat N, Oblinger MM, McCandless DW. Cytoprotective effect of estrogen on ammonium chloride-treated C6-glioma cells. Neurochem Res. 2004;29(7):1359–64. 23 Marti HH, Wenger RH, Rivas LA, Straumann U, Digicaylioglu M, Henn V, et al. Erythropoietin gene expression in human, monkey and murine brain. Eur J Neurosci. 1996;8:666–76. 24 Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R. A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J Biol Chem. 1994;269:19488–93. 25 Feinstein DL, Galea E, Roberts S, Berquist H, Wang H, Reis DJ. Induction of nitric oxide synthase in rat C6 glioma cells. J Neurochem. 1994;62:315–21. 26 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–9. 27 Mitchell DB, Acosta D. Evaluation of the cytotoxicity of tricyclic antidepressants in primary cultures of rat hepatocytes. J Toxic Environ Health. 1981;7:83–92. 28 Genc K. Erythropoietin and Parkinson’s disease: suggested mechanisms and therapeutic implications. Med Hypotheses. 2008;70:211–2. 29 Kumral A, Ozer E, Yilmaz O, Akhisaroglu M, Gokmen N, Duman N, et al. Neuroprotective effect of erythropoietin on hypoxic-ischemic brain injury in neonatal rats. Biol Neonate. 2003;83:224–8. 30 Iwasaki Y, Ikeda K, Ichikawa Y, Igarashi O, Iwamoto K, Kinoshita M. Protective effect of interleukin-3 and erythropoietin on motor neuron death after neonatal axotomy. Neurol Res. 2002;24:643–6. 31 Brines M, Cerami A. Emerging biological roles for erythropoietin in the nervous system. Nat Rev Neurosci. 2005;6: 484–94. 32 Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature. 2001;412:641–7. 33 Mitrovic B, Ignarro LJ, Montestruque S, Smoll A, Merrill JE. Nitric oxide as a potential pathological mechanism in demyelination: its differential effects on primary glial cells in vitro. Neuroscience. 1994;61:575–85. 34 Genc K, Genc S, Baskin H, Semin I. Erythropoietin decreases cytotoxicity and nitric oxide formation induced by inflammatory stimuli in rat oligodendrocytes. Physiol Res. 2006;55:33– 8. 35 Villa P, Bigini P, Mennini T, Agnello D, Laragione T, Cagnotto A, et al. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med. 2003;198:971–5. 36 Vairano M, Dello Russo C, Pozzoli G, Battaglia A, Scambia G, Tringali G, et al. Erythropoietin exerts anti-apoptotic effects on rat microglial cells in vitro. Eur J Neurosci. 2002;16:584–92. 37 Simenc J, Lipnik-Stangelj M. Staurosporine induces different cell death forms in cultured rat astrocytes. Radiol Oncol. 2012;46(4):312–20. 38 Shang Y, Wu Y, Yao S, Wang X, Feng D, Yang W. Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: involvement of PI3K/Akt and GSK-3 beta pathway. Apoptosis. 2007;12(12):2187–95. 39 Lotharius J, Dugan LL, O’Malley KL. Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons. J Neurosci. 1999;19(4):1284–93.
Kwon et al.
40 Dijkmans R, Van Damme J, Cornette F, Heremans H, Billiau A. Bacterial lipopolysaccharide potentiates gamma interferoninduced cytotoxicity for normal mouse and rat fibroblasts. Infect Immun. 1990;58(1):32–6. 41 Fisher JW. Erythropoietin: physiology and pharmacology update. Exp Biol Med (Maywood). 2003;228:1–14. 42 Nagai A, Nakagawa E, Choi HB, Hatori K, Kobayashi S, Kim SU. Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture. J Neuropathol Exp Neurol. 2001;60:386–92. 43 Bernaudin M, Bellail A, Marti HH, Yvon A, Vivien D, Duchatelle I, et al. Neurons and astrocytes express EPO
Cell protective effect of erythropoietin
mRNA: oxygen-sensing mechanisms that involve the redoxstate of the brain. Glia. 2000;30:271–8. 44 Sugawa M, Sakurai Y, Ishikawa-Ieda Y, Suzuki H, Asou H. Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation. Neurosci Res. 2002;44:391–403. 45 Chen ZY, Asavaritikrai P, Prchal JT, Noguchi CT. Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation. J Biol Chem. 2007;282:25875–83. 46 Kato S, Aoyama M, Kakita H, Hida H, Kato I, Ito T, et al. Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury. J Neurol Res. 2011;89:1566–74.
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