Biochemical and Biophysical Research Communications 372 (2008) 870874

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Biochemical and Biophysical Research Communications

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Hydrogen peroxide enhances TRAIL-induced cell death through up-regulation of DR5 in human astrocytic cells
Daeho Kwon a,*, Kyungsun Choi b, Chulhee Choi b, Etty N Benveniste c
a Medical Research Center for Environmental Toxico-Genomics and Proteomics, Korea University College of Medicine, Anam dong-5ga 126-1, Seongbuk-gu, Seoul 136-705, Republic of Korea b Department of Bio and Brain Engineering, KAIST, Daejeon, Republic of Korea c Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA

a r t i c l e

i n f o

a b s t r a c t
The central nervous system (CNS) is particularly vulnerable to reactive oxygen species (ROS), which have been implicated in the pathogenesis of various neurological disorders. The TNF superfamily of cytokines, especially tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces caspase-dependent cell death and is also implicated in various neurodegenerative diseases. In this study, we investigated the relationship between ROS and TRAIL-induced cell death. Exposure to hydrogen peroxide (H2O2) (100 lM) sensitized human astrocytic cells to TRAIL-induced cell death (up to 7-fold induction). To delineate the molecular mechanisms responsible for H2O2-induced sensitization, we examined expression of various genes (Caspase-8, Fas, FasL, DR4, DR5, DcR1, DcR2, TRAIL, TNFRp55) related to TRAIL-induced cell death. Treatment with H2O2 signicantly increased DR5 mRNA and protein expression in a time- and dose-dependent manner. H2O2-mediated cell death was blocked upon treatment with DR5:Fc protein, a TRAIL-specic antagonistic protein. These ndings collectively suggest that oxidative stress sensitizes human astroglial cells to TRAIL-induced cell death through up-regulation of DR5 expression. 2008 Elsevier Inc. All rights reserved.

Article history: Received 22 May 2008 Available online 4 June 2008

Keywords: TRAIL ROS DR5 Cell death Neuroimmunology Astrocytes

The generation of reactive oxygen species that include superoxide (O2), hydroxyl radical (OH), and hydrogen peroxide (H2O2) is related to cell death, stress-responses, aging, and various diseases such as cancer, ischemia/reperfusion injury, and atherosclerosis [14]. H2O2 and other ROS can induce cell death at relatively high concentrations in many cell types [59]. Recent observations suggest that lower concentrations of ROS are involved in inter-cellular communication and intra-cellular signaling in normal and pathological conditions [1013]. Superoxide is rapidly converted by non-enzymatic dismutation or superoxide dismutase (SOD) into H2O2 in vivo. Among the ROS, H2O2 is a ubiquitous molecule that is able to diffuse freely into membranes due to its nonpolar characteristics. H2O2 stimulates proliferation or enhances survival of a wide variety of cell types [1416]. Also, low concentrations of H2O2 stimulate endothelial migration as well as tube formation in an in vitro model of angiogenesis [17]. H2O2 initiates signals for mitochondrial translocation of Bax in lymphocytes [18]. H2O2 may be used as a second messenger in the NF-jB signaling system, despite its cytotoxicity [19,20]. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines. Mem* Corresponding author. Fax: +82 2 927 7220. E-mail address: dkwon@korea.ac.kr (D. Kwon). 0006-291X/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.148

bers of the TNF family contain highly conserved carboxyl-terminal domains and induce receptor trimerization to transduce intracellular signaling [21]. TRAIL can induce apoptotic cell death through caspase-dependent mechanisms [21,22]. TRAIL binds four different receptors, two of which, DR4 and DR5, induce apoptosis. However, decoy receptors for TRAIL, DcR1, and DcR2, do not have the intracytoplasmic death domain to transduce apoptotic death signals, and they protect cells from TRAIL-induced cell death by interfering with signaling through DR4 and DR5 [2224]. Astrocytes, the major glial cells in the central nervous system (CNS), maintain the homeostatic environment and also play an important role in immune regulation, acting as a source of chemokines, cytokines, and adhesion molecules [25]. Within the CNS, when stimulated by ROS, activated astrocytes can induce apoptosis-related gene expression [26,27]. In previous work, we demonstrated that H2O2 induces expression of Fas and FasL, and augments Fas-induced cell death by human astrocytoma cells [26]. In this study, we further investigated the possible immunomodulatory role of H2O2 in the CNS by determining its effects on expression of other apoptosis-related genes in astroglial cells. Our results indicate that treatment with H2O2 signicantly increased DR5 mRNA and protein expression, and H2O2-mediated DR5 expression augmented TRAIL-mediated cell death in human astrocytic cells.

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Materials and methods Cells and culture conditions. CRT-MG human astrocytoma cells were maintained in 10% FBS (GibcoBRL, Grand Island, NY, USA)RPMI 1640 (GibcoBRL) medium with 10 mM HEPES (pH 7.2) and 1 mM Earles balanced salt solution supplemented with 2 mM L-glutamine, 100 U/ml of penicillin, and 100 ug/ml of streptomycin, as previously described [28]. CH235-MG human astrocytoma cells were maintained in 10% FBSDMEM (GibcoBRL) medium, as previously described [28]. Human fetal primary astrocytes were kindly provided by Dr. In-Hong Choi (Yonsei University, Seoul, Korea) and were cultured in 10% FBSDMEM [29]. Reagents. H2O2 was purchased from Sigma (St. Louis, MO, USA). Mouse monoclonal anti-human DR5 antibody TRA-8 was a generous gift from Dr. Tong Zhou (University of Alabama at Birmingham, AL, USA) [30]. Human recombinant TRAIL (hrTRAIL) was puried as previously described [28] and was further puried by gel ltration chromatography. Goat anti-mouse immunoglobulin G1 (IgG1) antibody conjugated to phycoerythrin (PE) was purchased from Southern Biotechnology Associates (Birmingham, AL, USA). The TRAIL antagonistic protein TRAIL-R2:Fc was purchased from Alexis (San Diego, CA, USA). Detection of cell death. After treatment with TRA-8 or hrTRAIL, cells were washed twice with PBS, trypsinized, suspended in 200 ll of binding buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, 4% bovine serum albumin], and stained with 0.5 ng of Annexin V-uorescein isothiocyanate (FITC) and 2.5 ng of propidium iodide (PI) (BD Biosciences Clontec, Palo Alto, CA, USA). Ten thousand cells were analyzed with the FACStar within 30 min after staining. Cell death, including apoptosis and necrosis, was dened as cell fractions stained with Annexin V and/or PI. Total RNA isolation and RNase protection assay. Cells were washed with ice-cold phosphate buffered saline (PBS), and then RNA was isolated as previously described [31]. A linearized human apoptosis multiprobe set (hAPO-3c; PharMingen, San Diego, CA, USA) was in vitro transcribed with T7 RNA polymerase, resulting in antisense RNA probes. Ten micrograms of total RNA was hybridized with the hAPO-3c riboprobes. Values for mRNA levels were normalized to those for L32 mRNA levels for each experimental condition. Flow cytometric analysis. Astroglial cells (2 105 cells/well) were plated in six-well (35-mm2) plates (Costar, Cambridge, MA, USA) and grown to 90% conuency. For analysis of DR5 protein expression, cells were trypsinized, suspended in PBS containing 5% fetal bovine serum and 0.02% azide, incubated with TRA-8 (1 lg/ml), stained with PE-conjugated goat anti-mouse IgG antibody, washed twice, xed in 1% paraformaldehyde, and then analyzed by FACStar (BectonDickinson, Mountain View, CA, USA). Negative controls were incubated with an isotype-matched (IgG1) control antibody and stained with goat anti-mouse IgG antibody conjugated to PE. Ten thousand cells were analyzed for each sample. Electrophoretic mobility shift assay. Nuclear extracts from CH235MG cells incubated with TRAIL were prepared as previously described [31]. The nuclei were pelleted at 3000g for 10 min and resuspended in 200 ll of high salt buffer [10 mM HEPES, (pH 7.9), 1 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 400 mM NaCl, 15% glycerol, 1 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl uoride (PMSF), 1 mM sodium uoride, 10 lg/ml aprotinin, 10 lg/ml leupeptin, and 1 mM sodium orthovanadate]. The suspension was gently rocked for 30 min at 4 C followed by microcentrifugation at 12,000g for 10 min at 4 C. The protein concentration in the supernatant was determined with the Bradford method. Double stranded oligonucleotides containing consensus NF-jB sequences (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used in the EMSA. Oligonucleotides were end-labeled with [c-32P] ATP

(Dupont-NEN, Boston, MA, USA) using T4 polynucleotide kinase. Typically, 10 lg of nuclear extracts were equilibrated for 15 min in binding buffer [10 mM TrisHCl, (pH 8.0), 75 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.25 mM DTT] and 1 lg of poly dI/dC (Amersham Pharmacia Biotech, NJ, USA). 32P-labeled oligonucleotide probe (20,000 cpm) was added to the extracts and incubated for an additional 20 min at 4 C. Bound and unbound probes were then separated by electrophoresis on a 5% native polyacrylamide gel. Statistical analysis. Data are presented as means SD. Levels of signicance for comparisons between samples were determined using the Students t-test distribution. Statistical analyses between more than three samples were performed by ANOVA with Tukeys honest signicant difference post hoc test applied to signicant main effects or interactions (SPSS 12.0K for Windows, SPSS, Chicago, IL, USA). A p value <0.001 was considered signicant. Results and discussion We hypothesized that treatment with H2O2 might sensitize apoptosis-resistant astroglial cells to death receptor-mediated cell death by modulation of apoptosis-related gene expression. To address this, human CRT-MG astrocytoma cells were stimulated with TRAIL in the absence or presence of H2O2 for 24 h, and cell death was measured by ow cytometry after staining with annexin VFITC and PI. H2O2 alone did not induce cell death at the concentration of 100 lM. Treatment with TRAIL alone, at the concentration of 100 ng/ml, induced modest cell death ($10%) of CRT-MG cells. TRAIL-induced cell death was signicantly augmented by co-treatment with H2O2 (Fig. 1). Next, we analyzed apoptosis-related gene expression in CRT-MG and CH235-MG human astrocytoma cell lines after treatment with H2O2. Because CH235-MG cells are more resistant than CRT-MG cells to H2O2-induced cell death, we treated cells with different concentrations of H2O2. H2O2 induced mRNA expression of Fas and DR5 in a dose-dependent manner in CRT-MG cells (Fig. 2A), but did not affect expression of caspase-8 and TNFRp55. Up-regulation of Fas is shown as a positive control. In CH235-MG cells, H2O2 induced DR5 mRNA expression in time-dependent manner (Fig. 2B). Consistent with the mRNA results, H2O2 promoted DR5 protein expression by human astrocytoma cells (Fig. 2C). Human primary fetal astrocytes were also induced to express DR5 upon treatment with H2O2 (Fig. 2C). Since NF-jB is a pivotal transcription factor involved in innate immune responses [32], we further investigated whether NF-jB

100
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Fig. 1. H2O2 enhances TRAIL-induced cell death in human CRT-MG cells. Human CRT-MG cells were treated with H2O2 (50 or 100 lM) and/or TRAIL (100 ng/ml) for 24 h, and cell death was measured after staining with Annexin V-FITC and PI. P values indicate when the differences between groups were statistically signicant (Tukey post-hoc test applied to signicant effect of group ANOVA F5,24 = 155.8, P < 0.001).

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Fig. 2. H2O2 induces expression of apoptosis-related gene and DR5 protein in human astroglial cells. (A) CRT-MG cells were treated with varying doses of H2O2 (0100 lM) for 12 h, and total RNA was examined for Caspase-8, TNFRp55, Fas, DR5, and L32 mRNA by RPA. Fold induction of mRNA was normalized to the level of L32 mRNA expression. (B) CH235-MG cells were incubated with H2O2 (600 lM) for various time periods (024 h), and then total RNA was examined for Fas, DR5, and L32 mRNA by RPA. Fold induction of mRNA was normalized to the level of L32 mRNA expression. (C) CRT-MG, CH235-MG, and human fetal primary astrocytes were treated with H2O2 for 24 h. FACS analysis was performed for measurement of DR5 protein expression. Negative control was stained with isotype antibody conjugated with PE. Fold induction was calculated by dividing the uorescence intensity of H2O2-treated cells with that of untreated cells. Representative of three independent experiments.

was involved in H2O2-induced DR5 expression. CH235-MG cells were treated with H2O2 (600 lM) for various time periods, and nuclear extracts prepared and analyzed for NF-jB binding activity by EMSA. DNA binding activity of NF-jB was increased with H2O2 treatment in a time-dependent manner (13 h), and increased activity gradually returned to basal levels by 6 h (Fig. 3A). We next investigated whether H2O2-induced activation of NF-jB was necessary for inducing DR5 expression in human astroglial cells by using a pharmacological NF-jB inhibitor, MG-132 [33]. As shown in Fig. 3B, pretreatment with MG-132 completely suppressed H2O2-in-

A
NF-B

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Events

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Fig. 3. H2O2 induces activation of NF-jB, and inhibition of NF-jB abrogates H2O2induced DR5 expression. (A) CH235-MG cells were incubated in the absence or presence of H2O2 (600 lM) for varying time points (06 h), then nuclear extracts were prepared and analyzed for NF-jB binding activity by EMSA using the human consensus NF-jB probe. Bound and free probes were separated on 6% native polyacrylamide gels in 0.5 TBE. The data are representative of three independent experiments. (B) CH235-MG cells were pre-incubated with MG-132 (0.1 lM) for 1 h, then treated with H2O2 (600 lM) for 24 h, and then expression of DR5 measured by FACS. Representative of three independent experiments.

duced DR5 expression. These results indicate that activation of NF-jB is required for H2O2-induced DR5 expression in human astroglial cells. To address the biological function of H2O2-induced DR5 expression in human astroglial cells, cells were pre-incubated with TRAIL R2:Fc, a TRAIL antagonistic protein, for 1 h to block TRAIL-mediated death. Pre-incubation of astroglial cells with TRAIL R2:Fc blocked augmentation of TRAIL-induced cell death by H2O2 (Fig. 4A and B). Co-treatment with TRA-8, an agonistic anti-human DR5 antibody, and H2O2 induced cell death, which was also blocked by TRAIL R2:Fc (Fig. 4C). These results collectively indicate that H2O2 induces DR5 expression via NF-jB activation, and H2O2-induced DR5 expression augments TRAIL-mediated cell death in human astroglial cells. Stimulation of cells with cytokines, phorbol esters, or growth factors increases the secretion of H2O2 into the extracellular space in vitro [3436]. High concentrations of diffusible H2O2 have been implicated in cellular and tissue injury during pathologic conditions, for example, ischemia-reperfusion injury, hypoxia, and inammation, even though the molecular mechanism of its action has not yet been fully determined [37,38]. However, recent observations support an alternative role of H2O2:H2O2 alters cellular function by modulating cell signal transduction events in cells in vitro [3941]. In this study, we demonstrate that H2O2 induces DR5 expression in human astroglial cells through activation of the NF-jB transcription factor. We hypothesized that H2O2 could modulate apoptosis-related gene expression and as a result, affect immune modulation and neuronal degeneration in the CNS. To address this hypothesis, we used low concentrations of H2O2 that did not induce signicant cell death of the human astroglial cells. After treatment with H2O2, increased expression of DR5 was observed at both the mRNA and protein levels in human fetal primary astrocytes

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Fig. 4. H2O2 and TRAIL induce synergistic cell death, which is inhibited by DR5:Fc. CRT-MG cells (A) or CH235-MG cells (B) were pre-incubated for 1 h with DR5:Fc (100 ng/ ml), and then incubated in the absence or presence of TRAIL (100 ng/ml) for 24 h. CRT-MG and CH235-MG cells were treated with 100 lM and 600 lM of H2O2, respectively, for 24 h. P values indicate when the differences between groups were statistically signicant (Tukey post-hoc test applied to signicant effect of group (A)ANOVA F5,24=109.5, P < 0.001, (B) ANOVA F5,12=149.2). (C) CRT-MG cells were pre-incubated for 1 h with DR5:Fc (100 ng/ml), and incubated in the absence or presence of TRA8 (100 ng/ml) for 24 h. CRT-MG cells were treated with H2O2 (100 lM) for 24 h. Cell death was measured with Annexin V-FITC and PI. P values indicate when the differences between groups were statistically signicant (Tukey post-hoc test applied to signicant effect of group ANOVA F5,12=1514, P < 0.001).

and astrocytoma cells. There was a correlation between increasing doses of H2O2 and DR5 expression. The biological signicance of H2O2 was shown as enhanced cell death by either TRAIL or the TRA-8 antibody. Regulatory elements within the 50 anking sequence of the human DR5 gene have consensus sequences for transcription factors such as NF-jB and AP-1 [42]. Activation of NF-jB requires proteosomal degradation of IjB, which involves ubiquitination or other protein modications of IjB [43]. Since the proteosomal inhibitor MG-132 abrogated H2O2-induced NF-jB activation and subsequent induction of DR5 mRNA expression, proteosomal degradation of IjB seems to play an important role in H2O2-induced NF-jB activation and subsequent DR5 expression. In previous work, we observed that ROS induced Fas expression by human astroglial cells [26]. Collectively, H2O2 induces Fas and DR5 expression on human astroglial cells, and as a result, astrocytes become sensitized to treatment with TRAIL or FasL, leading to enhanced cell death. Our ndings suggest that DR5 expression may be induced by hypoxia/reperfusion and other forms of oxidative stress such as tumor-associated hypoxia. Another important aspect may be chemotherapeutic drug-induced apoptosis of tumor cells because many anticancer drugs produce ROS [4446]. Therefore, oxidative stress leading to up-regulation of DR5 plays a role in apoptosis in pathological conditions of the CNS. Acknowledgments This research was supported by the grant of Medical Research Center for Environmental Toxicogenomic and Proteomics (R13-

2003-016-00000-0), funded by Korea Science and Engineering Foundation and Ministry of Science and Technology. This work was also supported in part by NIH grants to E.N.B. References
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