amyloid-induced neuronal apoptosis - Springer Link

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Journal of Molecular Neuroscience Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN0895-8696/06/29:279–288/$30.00 JMN (Online)ISSN 1559-1166 DOI 10.1385/JMN/29:03:279

ORIGINAL ARTICLE

NF-κB-Associated MnSOD Induction Protects Against β-Amyloid-Induced Neuronal Apoptosis Pradoldej Sompol,1,2 Yong Xu,1 Wanida Ittarat,2 Chotiros Daosukho,1,2 and Daret St. Clair*,1 1Graduate

Center for Toxicology, University of Kentucky, Lexington, KY 40536; and 2Faculty of Medical Technology, Mahidol University, Bangkok, Thailand 10700 Received January 23, 2006; Accepted February 13, 2006

Abstract Expression of manganese superoxide dismutase (MnSOD), a nuclear-encoded mitochondrial primary antioxidant enzyme, is protective against various paradigms of oxidative stress–induced brain injury. We have shown previously that the presence of an intronic nuclear factor site, κB (NF-κB), in the MnSOD gene is essential for the induction of MnSOD by tumor necrosis factor α (TNF-α). However, whether activation of NF-κB is protective against oxidative stress–induced neuronal injury is unclear. In the present study, we demonstrate that TNF-α activates NF-κB activity in neuronal, SH-SY5Y, cells and preferentially enhances the binding of p50 and p65 to the promoter/enhancer regions of the MnSOD gene. Binding of NF-κB members to the MnSOD gene leads to the induction of MnSOD mRNA and protein levels. Consequently, induction of MnSOD by TNF-α primes neuronal cells to develop resistance against subsequent exposure to β-amyloid and FeSO4. Taken together, these results suggest that NF-κB might exert its protective function by induction of MnSOD leading to subsequent protection against oxidative stress–induced neuronal injury. DOI 10.1385/JMN/29:03:279 Index Entries: MnSOD; TNF-α; NF-κB; SH-SY5Y; apoptosis.

Introduction Manganese superoxide dismutase (MnSOD), a nuclear-encoded primary antioxidant enzyme, protects organisms against oxidative stress by rapidly dismutasing superoxide radicals to molecular oxygen and hydrogen peroxide (Weisiger and Fridovich, 1973). Hydrogen peroxide can be detoxified further by catalase or glutathione peroxidase (Michiels et al., 1994). Accumulating data suggest that MnSOD is critical for neuronal cell survival against oxidative stresses. MnSOD protects cortical neurons from Nmethyl-D-aspartate (NMDA) and nitric oxide–mediated neurotoxicity (Gonzalez-Zulueta et al., 1998).

Overexpression of MnSOD prevents cytotoxicity from Fe2+, β-amyloid, and nitric oxide-generating agents by attenuating nitrated proteins and 4-hydroxynonenal (4-HNE) levels (Keller et al., 1998). Overexpression of MnSOD reduces 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine toxicity (Klivenyi et al., 1998). Reduction of MnSOD in mouse cortical neurons significantly increases sensitivity to glutamate toxicity (Li et al., 1998). Crossing MnSOD heterozygous knockout mice into amyloid precursor protein mice increases β-amyloid levels and amyloid plaque burden in the brain (Li et al., 2004). Tumor necrosis factor α (TNF-α,) a proinflammatory cytokine, regulates cellular signaling,

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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280 including both proapoptotic and antiapoptotic pathways (Wajant et al., 2003). In the central nervous system (CNS), TNF-α is produced by neurons, microglia, and astrocytes at a low level (Vitkovic et al., 2000). It has been shown that the TNF-α level in cerebrospinal fluid (CSF) of Alzheimer’s patients can reach a level of 450 pg/mL, which is 25-fold higher than that in age-matched controls (Tarkowski et al., 1999). The increased levels of TNF-α in neurons have been suspected to protect against neuronal damage from cytotoxic insults, in part, via the induction of cytoprotective genes including MnSOD (Barger et al., 1995; Mattson et al., 1997; Bruce-Keller et al., 1999; Tamatani et al., 1999; Marchetti et al., 2004). The gene encoding human MnSOD consists of five exons interrupted by four introns (Wan et al., 1994; Zhu et al., 2001). The promoter region of the gene is 78% GC rich with no TATA or CAAT elements. The GC-rich promoter region contains multiple binding sites for the transcription factors SP-1 and AP-2 (Yeh et al., 1998; Zhu et al., 2001). Induction of the MnSOD gene is mediated via an intronic enhancer consisting of nuclear factor κB (NF-κB)-responsive elements located in the second intron of the gene (Xu et al., 1999). The redox-sensitive transcription factor NF-κB has been shown to be critical for neuronal cell survival, differentiation, and plasticity (Oneill and Kaltschmidt, 1997; Mattson and Camandola, 2001; Bhakar et al., 2002; Gutierrez et al., 2005). Although five members of the NF-κB family, including p65 (RelA), RelB, c-Rel, p52, and p50, are found in mammalian cells, p50 and p65 are the predominant family members found in nonhematopoietic cells. The p50p65 heterodimer, the predominant complex in many cell types, including neurons, is prevented from translocation into nucleus by IκB proteins (Karin and Lin, 2002). TNF-α stimulates the NF-κB signaling pathway through activation of IκB kinase (IKK), which phosphorylates IκB, leading to its subsequent ubiquitination and degradation by proteasome (Chen et al., 1995). Although study of brain injury paradigms indicates that lack of NF-κB activation enhances neuronal injury, it has been shown that activation of NF-κB promotes cell death in focal cerebral ischemia (Schneider et al., 1999). Thus, the role of NF-κB activation in neurons remains unclear. In the present study, we used immunoblot and electrophoretic mobility shift assay (EMSA) to show that TNF-α enhances the translocation of NF-κB into the nucleus and increases NF-κB-binding activity. Chromatin immunoprecipitation assay (ChIP) demonstrates that the p50-p65 complex binds to the


Sompol et al. promoter/enhancer element of the human MnSOD gene. Northern blot and RT-PCR indicate that TNF-α induces MnSOD gene expression. Immunoblot and the SOD activity gel further confirm that the TNF-α induced MnSOD mRNA was translated and processed into an authentic and active MnSOD protein. This study extends our previous studies of NF-κB-mediated transcriptional activation of the human MnSOD gene to neuronal cells and demonstrates that NF-κB activation plays a protective role against oxidative stress-induced neuronal apoptosis.

Materials and Methods Cell Culture and Apoptotic Cell Death Analysis SH-SY5Y, a human neuroblastoma cell, was grown in MEM, supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin/streptomycin/neomycin), 1% nonessential amino acid, and 1 mM sodium pyruvate. Cells were cultured at 37°C in a humidified incubator containing 5% CO2. The cells were treated with TNF-α (R&D System, Minneapolis, MN), β-amyloid 1–42 (Anaspec, San Jose, CA), and FeSO4 (Sigma, St. Louis, MO). Morphological assessment of apoptosis was performed after Hoechst staining. Cells were fixed in 4% paraformaldehyde for 5 min, rinsed in 1X PBS, and stained with 5 μg/mL Hoechst 33342 (Molecular Probe, Eugene, OR) for 10 min. The stained cells were viewed at excitation wavelength of UV filter by fluorescent microscopy (Olympus, Japan). The cell pictures were captured using Magnafire software. Intact, undamaged nuclei were identified as large and diffusely stained, whereas apoptotic nuclei exhibited condensed and fragmented chromatin. The apoptotic cells were counted in three random areas in triplicates in one treatment and analyzed statistically using three separate cell culture experiments.

Western Blot Analysis Whole-cell lysates or nuclear proteins were separated on a 12.5% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked and immunoblotted with primary antibodies, MnSOD (Upstate, Lake Placid, NY), p50 (Santa Cruz, Santa Cruz, CA), and p65 (Santa Cruz), followed by horseradish peroxidase–conjugated secondary antibodies (Santa Cruz). The signals were visualized by an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ). The membranes were then stripped and reprobed with antibodies, β-actin (Sigma), and G3PDH (Trevigen, Gaithersburg, MD), as internal controls.

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Inhibition of Neuronal Apoptosis by MnSOD Nuclear Protein Extraction and EMSA After TNF-α treatment, cells were rinsed twice in 1× PBS, resuspended, and incubated on ice for 10 min in three pellet volumes of buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT], and 0.2 mM PMSF). Nuclei were obtained by centrifugation at 14,000g and 4°C for 2 min and resuspended in two pellet volumes of buffer B (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 0.2 mM PMSF, and 1 µg/mL protease inhibitors [pepstatin, aprotinin, and leupeptin]). After incubation on ice for 20 min, the mixture was centrifuged at 12,000g and 4°C for 2 min. The supernatant was collected as nuclear proteins. Protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA), and the extracts were aliquoted and stored at –80°C. Double-stranded oligonucleotides corresponding to the consensus sequence of the NFκB-binding site (5′-GAGACTGGGGAATACCCCAGT-3′) were labeled with [γ-32P]ATP using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and purified on a 20% polyacrylamide gel. The nuclear extracts (20 µg) from SH-SY5Y cells were incubated on ice with 32P-labeled NF-κB probe for 30 min in a binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5 mM EDTA, 5% glycerol, 1 mM MgCl2, and 1 mM DTT). For supershift analysis, nuclear proteins were preincubated with 4 µg of antibody specific to p50 or p65 (Santa Cruz). DNA-protein complexes were separated from unbound probe on a 4% native polyacrylamide gel in Tris-borate/EDTA buffer (450 mM Tris-borate [pH 8.0] and 1 mM EDTA). After electrophoresis, the gels were dried and visualized using a Kodak phosphorimaging system.

Northern Blot and RT-PCR Total RNAwas isolated using Trizol reagent (Invitrogen, Carlsbad, CA). mRNAwas then purified using the Micro-FastTractΤΜ 2.0 kit (Invitrogen). Northern analysis was performed. Briefly, mRNA was separated on a 1.1% agarose/formaldehyde gel and then transferred to a nylon membrane. The membrane was baked at 80oC for 2 h, prehybridized at 42oC, and hybridized with 32P-labeled MnSOD cDNA. The membrane was exposed to Kodak film at –80oC, developed, stripped, and rehybridized with β-actin probe as an internal control. For RT-PCR, the mRNA was used in reverse transcription reaction SuperscriptΤΜ III First-Strand synthesis system (Invitrogen), according to the manufacturer’s instructions.


281 After digestion of the mRNA template by RNase H, cDNA was used as the template for RT-PCR using Taq DNA polymerase (Promega, Madison, WI) with a set of human MnSOD-specific primers. Sequences of the primers were: forward, 5′-AGCATGTTGAGAGCCGGGGCAGT-3′, and reverse, 5′-AGGTTGTTCACGTAGGCCGC-3′. Human β-actin was used for the housekeeping control, which was amplified with the following primers: forward, 5′-TGATGATATCGCCGCGCTCGTCGT-3′, and reverse, 5′CACAGCCTGGATAGCAACGTACAT-3′.

ChIP Assay Cells were grown to 70%–80% confluence and treated with 1 ng/mL TNF-α for 12 h. ChIP assay was performed using a ChIP-IT kit (Active Motif, Carlsbad, CA). Briefly, nuclear proteins were crosslinked to DNA by formaldehyde and stopped by glycine. The cells were suspended in lysis buffer, homogenized gently on ice using a glass Dounce homogenizer, and then centrifuged at 5000 rpm and 4°C for 10 min. The nuclei DNA were sheared using 200 U/mL enzymatic shearing cocktail from Enzymatic Shearing kit (Active Motif) at 37°C for 10 min to generate DNAfragments of 200–1000 bp in length; the semiquantitative amount of sample can be checked. Nonspecific background was removed by incubating the same amount of sheared chromatin with salmon sperm protein-G beads at 4°C for 4 h with agitation. The samples were centrifuged, and the recovered chromatins were incubated with 2 μg of p50 antibody at 4°C overnight with rotation. The DNA/protein/immune complexes were collected by adding protein-G beads and rotating at 4°C for 2 h. The beads were pelleted by centrifugation at 4°C for 2 min at 4000 rpm and washed sequentially following the kit protocol. The pulled-down DNA was eluted in elution buffer (1 M NaHCO3 and 1% SDS). The DNA was reverse cross-linked with 5 M NaCl, and RNA was removed using RNase A. Protein contamination was removed using 0.5 M EDTA, 1 M Tris-HCl (pH 6.5), and proteinase K solution. DNA was purified in minicolumns. To quantify the GCrich MnSOD promoter fragment, an AccuPrimer GCRich DNA polymerase (Invitrogen) was used with 28 cycles at 95°C for 45 s, 60°C for 30 s, and 72°C for 30 s. The promoter region (–127 to –6) was amplified using a pair of primers: upper-strand primer, 5′ACAGGCACGCAGGGCACCCCCGGGGTT-3′, and lower-strand primer, 5′- TCCTGCGCCGCCCGCGGGCCTTAAGAAA-3′. Taq DNA polymerase (Promega) was used to amplify the enhancer fragment

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Fig. 1. Detection of cytotoxicity of oxidative stress–inducing agents to neuronal cells. Cells were pretreated with TNF-α at indicated concentrations followed by saline or pretreated with saline and followed by 10 μM β-amyloid 1–42 or 0.5 mM FeSO4 for 24 h. (A) Representative pictures show increased apoptotic cells in β-amyloid 1–42 or FeSO4-treated cells. (B) Quantitative analysis of apoptotic cells from three independent experiments, each with triplicate.

(1912–2083) with 25 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The sequence of primer sets was: upper strand, 5′-CGGGGTTATGAAATTTGTTGAGTA-3′, and lower strand, 5′-CCACAAGTAAAGGACTGAAATTAA-3′. The standard semiquantitative PCR procedure with 20, 25, 28, and 30 cycles was performed. The PCR products from both 25 and 28 cycles showed significant difference between TNF-α treatment and nontreatment. To detect p50-p65 interaction in chromatin, the p50 antibody pulled-down chromatin complexes were separated by SDS-PAGE and immunoblotted with p65 antibody.

SOD Activity Gels The cells were harvested, frozen, and thawed in 50 mM potassium phosphate buffer (pH 7.8). Protein (400 μg) was electrophoresed on a nondenatured polyacrylamide gel consisting of a 5% stacking gel and a 12.5% separating gel at 4oC. The gel was stained in 2.43 mM NBT for 20 min and followed by 0.028 mM riboflavin and 280 mM N,N,N9,N9-tetramethylethylenediamine in 50 mM potassium phosphate buffer (pH 7.8) for 15 min in the dark. The achromatic bands of enzyme activity were visualized


by exposing the stained gel to fluorescent light until the clear zone of enzyme activity appeared on a dark purple background.

Results and Discussion TNF-α Reduces β-amyloid or FeSO4-Induced Neuronal Cell Death TNF-α is expressed in neurons, microglia, and astrocytes at a low level in normal brain (Vitkovic et al., 2000). Increased TNF-α levels were found in the CSF of Alzheimer’s disease (AD) (Tarkowski et al., 1999). TNF-α has been shown to modulate both proapoptotic and antiapoptotic signaling in different cell types (Szelenyi, 2001; Wajant et al., 2003; Varfolomeev and Ashkenazi, 2004). It has been demonstrated that TNF-α protects neurons from various toxic insults (Barger et al., 1995; Houzen et al., 1997; Mattson et al., 1997; Bruce-Keller et al., 1999; Kaltschmidt et al., 1999; Tamatani et al., 1999; Glazner and Mattson, 2000; Diem et al., 2001; Viel et al., 2001). However, TNF-α has also been shown to inhibit neuronal cell growth and mediate cell death (Kenchappa et al., 2004). The differential effect of TNF-α in neurons is thought to depend on culture age, culture

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Fig. 2. Pretreatment with TNF-α protected against subsequent oxidative stress–induced cell death. Cells were pretreated with TNF-α for 24 h, followed by treatment with 10 μM β-amyloid 1–42 or 0.5 mM FeSO4 for 24 h. Apoptotic cells were quantified as described in Fig. 1. Values are the mean ± S.E.M. of three independent experiments. (*) p < 0.01; (**) p < 0.001; ANOVA with Turkey’s multiple comparison.

density, and the presence of glial contamination in the primary cell cultures. To investigate the effect of TNFα on pure neurons, SH-SY5Ycells were pretreated with various concentrations of TNF-α (0.05, 0.5, and 1 ng/mL) for 24 h prior to exposure to 10 μM β-amyloid 1–42 or 0.5 mM FeSO4 for 24 h. Apoptotic cell death was estimated by analysis of nuclear DNA condensation and fragmentation after Hoechst 33342 staining (Fig. 1A). Control groups treated with saline for 24 h, followed by saline for 24 h or TNF-α for 24 h, followed by saline for 24 h, had no significant cell death (Fig. 1B). In contrast, exposure of cells to saline for 24 h followed by β-amyloid and FeSO4 resulted in approx 20-fold neuronal cell death compared with control groups (Fig. 1B). When cells were pretreated with TNF-α and followed with β-amyloid or FeSO4, neuronal cell death was reduced significantly (Fig. 2A,B). Our results demonstrating that treatments with low doses of TNF-α have no effect on neuronal cell death are consistent with the results of previous studies using doses of TNF-α up to 100 ng/mL (Tamatani et al., 1999; Combs et al., 2001; Floden et al., 2005). However, TNF-α increased cell death when combined


with toxic insults such as β-amyloid, NMDA, and glutamate (Stepanichev et al., 2003; Floden et al., 2005; Zou and Crews, 2005). These results indicate that TNF-α plays a dual role on proapoptotic and antiapoptotic pathways depending on the mode and sequence of exposure. Our data, which demonstrate that TNF-α itself has no effect on neuronal cell death but does provide adaptive response against subsequent toxic insults, indicate that TNF-α can play a role in the priming and desensitizing effects on neuronal cells.

TNF-α Enhances NF-κB Activation in Neuronal Cells Nuclear factor κB (NF-κB), a redox-sensitive transcription factor, is known to play a protective role in many tissues in response to stress, inflammation, and immunity. NF-κB activation is required for CNS development and survival (Bhakar et al., 2002). NFκB signaling is essential for neurite growth. Blocking of NF-κB caused reductions in total neurite length, total branch number, alteration of synaptic plasticity, and long-term depression of synaptic transmission in cortical and hippocampal cultures (Albensi

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Fig. 3. Analysis of nuclear p50 and p65 levels. (A) Representative immunoblot showing TNF-α increases p50 and p65 in the nucleus. Quantitative analysis of p50 (B) and p65 (C) levels in nucleus from three independent cell culture experiments. (*) p < 0.05, ANOVA with Turkey’s multiple comparison. (D) Representative EMSA shows detection of NF-κBbinding activity. Arrows indicate positions of NF-κB complex and p50- and p65-supershifted bands.

Fig. 4. NF-κB-mediated TNF-α-induced MnSOD expression. (A) Representative ChIP assay from two independent experiments demonstrating p50 pulled-down enhancer and promoter regions of the human MnSOD gene. (B) Representative Western blot demonstrating that p65 was present in the ChIP complex.


and Mattson, 2000; Foehr et al., 2000; Gutierrez et al., 2005). To determine the effect of TNF-α on NF-κB activation in neuronal cells, nuclear extracts were prepared and analyzed by Western blot. TNF-α treatment increased nuclear levels of p50 and p65 in a time-dependent manner (Fig. 3A–C), suggesting that TNF-α was able to increase NF-κB components in the nucleus. EMSA was performed to examine binding activity using a probe containing NF-κB consensus sequence. The results shown in Fig. 3D indicate that TNF-α significantly increased NF-κB-binding activity through increasing levels of p50 and p65 in the nucleus. These results are consistent with results from previous studies, which demonstrated that TNF-α activates NF-κB signaling pathway through binding to its receptors leading to activation of IKK. The activated IKK phosphorylates an inhibitory subunit, IκB, resulting in IκB degradation allowing NF-κB translocation into the nucleus and subsequently regulating NF-κB target genes (Mattson et al., 2000; Bui et al.,

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Fig. 5. TNF-α increased MnSOD transcripts in neuronal cells. Representative Northern blotting shows MnSOD transcripts (A) and repeat with quantitated RT-PCR using two sets of independent cell cultures (B).

2001). Our results also suggest that at selected concentrations of TNF-α activation of NF-κB might lead to increased transcription of its cytoprotective target genes.

TNF-α−Mediated Activation of NF-κB Pathway Leads to Increased MnSOD Gene Expression in Neuronal Cells It has been documented that levels of oxidativemodified proteins, lipids, and DNA are increased but levels of antioxidant enzymes, including SOD and catalase, are markedly reduced in AD (Lyras et al., 1997; Marcus et al., 1998; Gibson et al., 2005; Wang et al., 2005). Depletion of antioxidant enzymes is consistent with mitochondrial abnormalities and dysfunction leading to neuronal apoptosis (Hirai et al., 2001; Sullivan and Brown, 2005; Takuma et al., 2005). Reduction of MnSOD enhances sensitivity to glutamate-induced excitotoxicity (Li et al., 1998), and MnSOD-deficient mice have increased β-amyloid protein and plaque burden in the brain (Li et al., 2004). Overexpression of MnSOD has been shown to prevent neuronal cell death by suppressing nitric oxide–mediated neurotoxicity, peroxynitrite production, lipid peroxidation, and increased mitochondrial function (Gonzalez-Zulueta et al., 1998; Keller et al., 1998; Klivenyi et al., 1998). To determine whether TNF-α-mediated NF-κB activation leads to increased binding of NF-κB members to the promoter/enhancer of the MnSOD gene in neurons, we applied ChIP assay to quantify the binding of NF-κB to the transcriptional regulation region of the human MnSOD gene. Chromatin complex from TNF-α-treated SH-SY5Y cells was precipitated


by a p50 antibody. The resulting DNA/protein complex was deproteinated and used as the template for PCR amplification. Quantitative PCR showed that both enhancer and promoter regions of the human MnSOD gene were increased significantly in TNF-α-treated cells as compared with untreated controls (Fig. 4A). Western blot analysis showed that the p50 partner, p65, also increased in p50-precipitated chromatin (Fig. 4B). These results are consistent with those from supershift experiments (Fig. 3D) identifying p50 and p65 as members of the NFκB family that are predominantly present in neuronal cells. To determine whether the occupancy of NF-κB in the promoter/enhancer leads to the transcription of the MnSOD gene, mRNA was prepared for Northern blot analysis and RT-PCR. As shown in Fig. 5A, TNF-α significantly increased endogenous MnSOD mRNA levels observed in 1.0-, 4.0-, and 6.0-kb MnSOD transcripts. Consistently, RTPCR results also showed an increase in MnSOD mRNA levels after 12 h TNF-α treatment compared with untreated control (Fig. 5B). Finally, to determine whether TNF-α−induced MnSOD mRNA was translated into authentic MnSOD protein, total cellular proteins were extracted to detect MnSOD protein levels and MnSOD activity. Western blotting analysis showed that after treatment with TNF-α, the protein levels of MnSOD were increased in a dose-dependent manner (Fig. 6A). The SOD activity gel indicated that MnSOD activity was also increased in the same pattern shown in immunoblots (Fig. 6B). Taken together, these results suggest that TNF-α induces MnSOD gene transcription in neuronal cells by increasing NF-κB proteins binding to

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Fig. 6. TNF-α increased MnSOD protein and activity levels. (A) Representative Western blots demonstrate TNF-α increases MnSOD protein levels. (B) Activity gels demonstrating the presence of enzymatically active MnSOD. Numbers above each lane indicate the ratio of MnSOD protein and MnSOD activity (A and B, respectively).

MnSOD promoter/enhancer regions leading to an enhanced expression of active MnSOD protein. These results are the first to directly demonstrate that increased p50/p65 occupancy at the promoter/enhancer of its target genes might determine whether activation of NF-κB is cytoprotective or cytotoxic to neuronal cells. Our results also demonstrated that prior exposure to TNF-α had a priming effect that might prevent neuronal injury from subsequent exposure to oxidative stress–inducing insults.

Acknowledgments This work was supported by NIH grants CA049797 and AG05119 to D. S. and by the Royal Golden Jubilee Research Fellowship (The Thailand Research Fund) to P. S. We thank Dr. Jeffrey N. Keller for the SH-SY5Y cells and Edgardo R. Dimayuga for his assistance with the SH-SY5Y cells.

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