Induction of Pi Form of Glutathione S-Transferase by Carnosic Acid Is

Sep 30, 2014 - ... GSTM, and GSTP are expressed in the central nervous system. ...... J., Ketterer , B., and Taylor , J. B. (1996) The organization of...
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Induction of Pi Form of Glutathione S‑Transferase by Carnosic Acid Is Mediated through PI3K/Akt/NF-κB Pathway and Protects against Neurotoxicity Chia-Yuan Lin,† Jing-Hsien Chen,‡,§ Ru-Huei Fu,∥,⊥ and Chia-Wen Tsai*,† †

Department of Nutrition, China Medical University, 40402 Taichung, Taiwan School of Nutrition, Chung Shan Medical University, 40201 Taichung, Taiwan § Department of Medical Research, Chung Shan Medical University Hospital, 40201 Taichung, Taiwan ∥ Graduate Institute of Immunology, China Medical University, 40402 Taichung, Taiwan ⊥ Center for Neuropsychiatry, China Medical University Hospital, 40402 Taichung, Taiwan ‡

ABSTRACT: Carnosic acid (CA), a diterpene found in the rosemary (Rosmarinus officinalis), has been reported to have a neuroprotective effect. Glutathione S-transferase (GST) P (GSTP) is a phase II detoxifying enzyme that provides a neuroprotective effect. The aim of this study was to explore whether the neuroprotective effect of CA is via an upregulation of GSTP expression and the possible signaling pathways involved. SH-SY5Y cells were pretreated with 1 μM CA followed by treatment with 100 μM 6hydroxydopamine (6-OHDA). Both immunoblotting and enzyme activity results show that CA also induced protein expression and enzyme activity of GSTP. Moreover, CA significantly increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt, the nuclear translocation of p65, but not mitogen-activated protein kinases (p < 0.05). Pretreatment with LY294002 (a PI3K/Akt inhibitor) suppressed the CA-induced phosphorylation of IκB kinase (IKK) and IκBα, p65 nuclear translocation, and nuclear factor-kappa B (NF-κB)-DNA binding activity as well as GSTP protein expression. Furthermore, CA attenuated 6-OHDA-induced caspase 3 activation, and cell death was reversed by GSTP siRNA or LY294002 treatment. Additionally, male Wistar rats with lesions induced by 6-OHDA treatment in the right striatum responded to treatment with CA, which significantly reversed the reduction in GSTP protein expression that resulted from lesioning. We suggest that CA prevents 6-OHDA-induced apoptosis through an increase in GSTP expression via activation of the PI3K/Akt/ NF-κB pathway. Therefore, CA may be a promising candidate for use in the prevention of Parkinson’s disease.

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INTRODUCTION Parkinson’s disease is a progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra.1,2 The mechanisms of dopaminergic neuron death in Parkinson’s disease are still unclear, although some studies have indicated that oxidative stress may play an important role. The causes of Parkinson’s disease are related to the formation of lipid peroxidation products, the depletion of reduced glutathione, and a deficit of mitochondrial complex I.3 Accumulating evidence supports that free radical scavenging or induction of antioxidant enzymes by bioactive plant compounds could protect dopaminergic neurons against oxidative damage.4−6 Glutathione S-transferase (GST) catalyzes the conjugation of glutathione with a variety of electrophilic xenobiotics and facilitates their excretion. This enzyme also removes hydrogen peroxide and reduces the lipid hydroperoxides.7 The cytosolic GSTs in human tissues are divided into seven classes: A (alpha), M (mu), O (omega), P (pi), S (sigma), T (theta), and Z (zeta).8 The pi form of GST (GSTP) is highly expressed in glial cells and in dopaminergic neurons of the substantia nigra.9,10 Studies in humans have shown that polymorphisms of © XXXX American Chemical Society

GSTP may increase susceptibility to Parkinson’s disease after pesticide exposure.11 In mice, Castro-Caldas et al.12 suggested that GSTP knockout mice were more susceptible to the neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) than were C57BL/6 wild type mice. In rat pheochromocytoma (PC12) cells, knockdown of GSTP expression exacerbates dopamine-induced apoptosis.13 By contrast, overexpression of GSTP in cultured cortical neuronal cells decreases rotenone-induced neurotoxicity and leads to reduced oxidative stress and endoplasmic reticulum stress.11 Thus, these results suggest that the neuroprotection of GSTP may provide a new approach to delay Parkinson’s disease progression. The inducibility of GSTP is regulated by multiple factors, including activator protein-1 and nuclear factor-kappa B (NFκB).14,15 NF-κB plays important roles in regulating the expression of genes involved in a variety of cellular processes, including cell survival, differentiation, and inflammation.16 Recently, the NF-κB pathway has garnered much attention Received: July 25, 2014

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(protocol no. 97-140-N). The animals were acclimatized for 7 days prior to study use. Rats were randomly divided into three groups. Group 1 was a sham group and received 2.5 μL of 0.5% ascorbic acid in saline (n = 10). Group 2 was a lesion group and was treated with 6-OHDA (12.5 μg/2.5 μL; 0.5% ascorbic acid in saline) in the right striatum (n = 11). Group 3 was administered CA (20 mg/kg body weight orally) (n = 11) three times each week for 3 weeks. CA was suspended in 0.5% carboxymethylcellulose sodium. 6-OHDA was administered as a single injection in the right striatum on day 22. The animals were sacrificed after 14 days of lesioning, and the striatum was quickly dissected out for the Western blot assay. Intrastriatal Administration of 6-OHDA. The rats were anesthetized with Zoletil intraperitoneally. After the rats were anesthetized, the head of the rat was mounted in a stereotaxic apparatus frame. The skin was cut to expose the skull. The lesion coordinates used were as follows: anteroposterior (AP) = +1.5 mm; lateral (L) = −4 mm; dorsoventral (V) = −7.2 mm. 6-OHDA was dissolved in sterile 0.5% ascorbic acid at a concentration of 12.5 μg/2.5 μL and was administrated in the right striatum (1 μL/min). The needle was kept in place for an additional 1 min before being slowly retracted. As indicated above, the sham-operated rats received 2.5 μL of 0.5% ascorbic acid in saline. Cell Culture and Treatment. SH-SY5Y cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). The method is described in our previous study.28 The cells were maintained at 37 °C in the incubator with a humidified atmosphere of 5% CO2 and were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM Lglutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acid, 1.0 mM sodium pyruvate, 1 × 105 unit/L penicillin, and 100 mg/L streptomycin. For all studies, SH-SY5Y cells were seeded on 35 mm plastic tissue dishes (Corning, NY) at a density of 1.2 × 106 cells per dish and were treated once the cells reached 80% confluence. CA and 6-OHDA were dissolved in DMSO. Cells were pretreated with CA for 18 h followed by treatment with 100 μM 6-OHDA for 12 or 18 h. In the inhibition of kinase experiment, PI3K/Akt inhibitor (LY294002) at a concentration of 5 μM was added 1 h before CA treatment. The control cells were treated with 0.3% DMSO alone. Cell Viability Assay. SH-SY5Y cells were cultured with 1 μM of CA for 18 h and were then stimulated with 100 μM 6OHDA for the indicated time. Cell viability was examined by using the MTT assay as previously described.28 Briefly, cells were washed with phosphate-buffered saline and cultured in MTT solution (5 mg/mL) at 37 °C for 2 h. After the MTT solution was removed, the formazan product was dissolved in isopropanol. Absorbance was detected at 570 nm by use of a microplate reader (Bio Rad, Japan). Western Blot Assay. The method was determined by our previous study.29 After treatment, cells were washed with cold phosphate-buffered saline and were then harvested in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and phosphatase inhibitor). Lysates were centrifuged at 14 000g for 20 min at 4 °C. Protein concentrations were measured with a Coomassie plus protein assay reagent kit (Pierce, Rockford, IL). Cell lysates were applied to 7.5−12.5% SDS-PAGE gels and were electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The nonspecific

for its critical role in neuronal survival and protection from toxic insults.17,18 For example, Maggirwar et al.19 suggested that nerve growth factor promotes the survival of sympathetic neurons by activating NF-κB. Furthermore, in mice lacking NFκB, administration of the excitotoxin kainite causes hippocampal pyramidal neuron injury.20 The NF-κB family comprises five related members: RelA (p65), c-Rel, RelB, NF-κB1(p105)/ p50, and NF-κB2 (p100)/p52.21 The NF-κB complex, which mainly consists of a p65/p50 heterodimer, is retained in the cytoplasm with inhibitory protein, IκB. In response to stimuli, the IκB kinase (IKK) complex phosphorylates IκB protein with subsequent degradation by the 26S proteasome. The released NF-κB heterodimer translocates to the nucleus, where it regulates gene expression.21 Studies have suggested that the phosphatidylinositide 3-kinase (PI3K)/protein kinase (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways are a common signal mediating NF-κB activity.22,23 For example, Kim et al.23 indicated that metallothionein-III protects neuronal cells from hydrogen peroxide- and doxorubicin-induced neurotoxicity by activating NF-κB via the TrkA receptor tyrosine kinase/PI3K/Akt signaling pathway Carnosic acid (CA) is a diterpene from rosemary (Rosmarinus officinalis), and it has been reported to possess diverse biological properties, including antioxidative, neuroprotective, and anticarcinogenic activity.24−26 However, the mechanisms of neuroprotection by CA are not fully understood. Evidence shows that CA attenuates dieldrin-induced cell death by enhancing brain-derived neurotrophic factor in SN4741 dopaminergic neuronal cells.27 Another study suggested that CA protects the brain and cortical neurons from middle cerebral artery occlusion/reperfusion injury and glutamate, respectively.26 Our previous study indicated that the prevention of 6-hydroxydopamine (6-OHDA)-induced oxidative stress and apoptosis by CA is related to glutathione synthesis in SH-SY5Y neuronal cells.28 We were interested in further examining whether the neuroprotection of CA is via an upregulation of GSTP expression and the possible signaling pathways involved. Therefore, in the present study, we examined the modulatory effect of CA on GSTP expression in SH-SY5Y cells. Moreover, we determined the signaling pathway involved in the neuroprotective effect of GSTP.

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MATERIAL AND METHODS Materials. CA, 6-OHDA, sodium bicarbonate, sodium pyruvate, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, Tween 20, LY294002, and ethacrynic acid were purchased from Sigma Chemical Company (St. Louis, MO). DMEM, Lglutamine, nonessential amino acid, trypsin-EDTA, and penicillin−streptomycin solution were obtained from Gibco Laboratory (Gaithersburg, MD). Fetal bovine serum was purchased from Hyclone (Logan, UT). Glycine, acrylamide, and Tris were purchased from U.S. Biological (Swampscott, MA). Animals and Treatments. Male Wistar rats (approximately 6−8 weeks old) were used in this study and were purchased from BioLASCO Experimental Animal Center (Taipei, Taiwan). Rats were housed in a temperature-controlled room at 23 ± 1 °C with a 12 h light/dark cycle. Animals were housed in groups of four animals per cage with free access to chow diet and water ad libitum. For the use of animals in the study, ethical approval was obtained from the Institutional Animal Care and Use Committee of China Medical University B

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binding sites on the membranes were blocked at 4 °C overnight with 50 g/L nonfat dry milk. The blots were then incubated with primary antibodies against caspase 3, cleaved caspase 3, PARP, p38, Akt, phospho-p38, phospho-Akt, phospho-IKKα/β, or phospho-IκBα (all purchased from Cell Signaling Technology, Beverly, MA); β-tubulin (purchased from Sigma Chemical Company, St. Louis, MO); JNK1, ERK1/2, phospho-JNK1, phospho-ERK1/2, IKKα/β, or IκBα (all from Santa Cruz Biotechnology, Santa Cruz, CA); GSTA and GSTM (purchased from Oxford Biomedical Research, Oxford, MI); GSTP (purchased from Transduction Laboratories, Lexington, KY); and p65 (purchased from BD Bioscience, San Jose, CA) overnight at 4 °C and were subsequently incubated with horseradish peroxidase-conjugated goat antirabbit IgG or goat antimouse IgG (all purchased from PerkinElmer life Sciences, Boston, MA) and rabbit antigoat IgG (purchased from R&D Systems Inc., Minneapolis, MN). The bands were detected by using an enhanced chemiluminescence kit (purchased from PerkinElmer Life Science, Boston, MA). Enzyme Activity Assay. The enzyme activity of GSTP was measured by using the conjugation of glutathione and ethacrynic acid as previously described.30 Briefly, the reaction mixture in a final volume of 1 mL contained 100 mM phosphate buffer, 1 mM glutathione, 20 mM ethacrynic acid, and an appropriate amount of the total protein. The ethacrynic acid conjugated form was measured at 270 nm. Transient Transfection of Small RNA Interference (siRNA). In briefly, SH-SY5Y cells were transfected with nontargeting control small interfering RNA (siRNA) or human GSTP siRNA (50 nM) (all from MDBio, Taipei, Taiwan) by using the Dharma FECT siRNA transfection reagent according to the manufacturer’s protocol (Thermo Fisher Scientific) for 24 h. The sequence of the human GSTP siRNA was as follows: 5′-GCUGAUCCAUGAGGUCCUATT-3′. Twenty-four hours after transfection, the cells were pretreated with 1 μM CA and were then stimulated with 100 μM 6-OHDA for 12 or 18 h. The protein expression was examined by Western blot analysis, and the cell viability was determined by MTT assay. Preparation of Nuclear Extract. Nuclear protein was prepared as previously described.28 SH-SY5Y cells were exposed to CA for the indicated times and were then washed in cold phosphate-buffered saline followed by scraping from the dishes with phosphate-buffered saline. Cell homogenates were centrifuged at 2000g for 5 min. The cell pellets were allowed to swell on ice for 15 min after the addition of 200 mL of hypotonic buffer containing 10 mM HEPES, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5% Nonidet P-40, 4 mg/L leupeptin, 20 mg/L aprotinin, and 0.2 mM phenylmethylsulfonyl fluoride. After centrifugation at 6000g for 15 min, pellets containing crude nuclei were resuspended in 40 μL of hypertonic buffer containing 10 mM HEPES, 400 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 4 mg/L leupeptin, 20 mg/L aprotinin, and 0.2 mM phenylmethylsulfonyl fluoride and were incubated for an additional 30 min on ice. The nuclear extracts were then obtained by centrifugation at 10 000g for 15 min and were frozen at −80 °C. Electromobility Gel Shift Assay (EMSA). The EMSA method described by Tsai et al.29 was used to determine NF-κB nuclear protein−DNA binding activity. The LightShift Chemiluminescent EMSA kit (Pierce Chemical) and synthetic biotin-labeled double-stranded human GSTP NF-κB oligonucleotide (forward: 5′-TCTTAGGGAATTTCCCCCCGCGA-

3′; reverse: 5′-TCGCGGGGGGAAATTCCCTAAGA-3′) were used to measure the effect of CA on NF-κB nuclear protein− DNA binding activity. Unlabeled double-stranded NF-κB (200 ng) was also used to confirm specific binding. Six micrograms of nuclear protein, poly(dI-dC), and biotin-labeled doublestranded human GSTP NF-κB oligonucleotide were mixed with the binding buffer to a final volume of 20 μL and were incubated at room temperature for 30 min. The nuclear protein−DNA complex was separated by electrophoresis on a 6% Tris-boric acid-EDTA-polyacrylamide gel and was then electrotransferred to a Hybond-N+ nylon membrane (GE Healthcare, Buckinghamshire, United Kingdom). The membrane was incubated with streptavidin-horseradish peroxidase, and the nuclear protein−DNA bands were developed by using an enhanced chemiluminescence kit. Statistical Analysis. Statistical analysis was performed with commercially available software (SAS Institute Inc., Cary, NC). Data were analyzed by means of one-way ANOVA, and the significant difference among treatment means was assessed by use of Tukey’s test. A value of p < 0.05 was considered to be significant.

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RESULTS CA Increased GSTP Protein Expression and Enzyme Activity. Both immunoblotting and enzyme activity results shown that CA dose-dependently increased the protein expression and enzyme activity of GSTP (Figure 1A and B). CA at 1 μM caused a 2.7- and a 1.5-fold increase in the GSTP protein level and enzyme activity compared with that in the control group, respectively (p < 0.05). However, CA had no significant effect on the protein level of GSTA or GSTM.

Figure 1. CA induced the protein expression and enzyme activity of GSTP in SH-SY5Y cells. (A) Cells were treated with various concentration of CA for 18 h. GST family protein expression was determined by Western blotting. β-Tubulin was used as the loading control. (B) The enzyme activity of GSTP was measured by using ethacrynic acid as a substrate. The level in control cells was set at 1.0. One representative immunoblot out of three independent experiments is shown. Values are means ± SD, n = 3. Means without a common letter differ, p < 0.05. C

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CA Activated the PI3K/Akt/NF-κB Pathway. The p65 nuclear translocation was activated in cells treated with CA for 3 to 6 h (Figure 2A). Moreover, CA increased the

Figure 3. CA activated the PI3K/Akt pathway. SH-SY5Y cells were treated with 1 μM CA for 15, 30, or 60 min. The phosphorylation and total expression of Akt and MAPK proteins were determined by Western blotting. β-Tubulin was used as the loading control. The level in control cells was set at 1.0. One representative immunoblot out of three independent experiments is shown. Values are means ± SD, n = 3. Means without a common letter differ, p < 0.05. Figure 2. CA activated the NF-κB pathway. SH-SY5Y cells were incubated with 1 μM CA for the indicated times. The protein expression of nuclear p65 (A) and the phosphorylation and total expression of IKKα/β and IκBα protein (B) were determined by Western blotting. β-Tubulin and PARP were used as the loading controls. The level in control cells was set at 1.0. One representative immunoblot out of three independent experiments is shown. Values are means ± SD, n = 3. Means without a common letter differ, p < 0.05.

double-stranded oligonucleotide (cold) (Figure 4B). These results suggested that CA up-regulated the protein expression of GSTP and NF-κB activation through the PI3K/Akt pathway. LY294002 or GSTP siRNA Attenuated the Neuroprotective Effect of CA. LY294002 and GSTP siRNA were used to demonstrate that the Akt-GSTP pathway is crucial for the neuroprotective effect of CA. The immunoblotting results showed that CA suppressed the 6-OHDA-increased ratio of cleaved caspase 3 and caspase 3. This effect was reversed in cells treated with LY294002 (Figure 5A). Moreover, LY294002 reduced the ability of CA to reverse the inhibition of GSTP protein and cell viability by 6-OHDA (Figure 5A and B). To further confirm these observations, cells were transfected with GSTP siRNA. After transfection for 24 h, the induction of GSTP protein by CA was decreased (Figure 6A). As expected, GSTP siRNA reversed the ability of CA to suppress the 6OHDA-induced ratio of cleaved caspase 3 and caspase 3 (Figure 6B). In addition, the ability of CA to ameliorate the reduction in GSTP protein expression and in cell viability induced by 6-OHDA was decreased in the presence of GSTP siRNA (Figure 6B and C). These results suggested that the Akt/GSTP pathway was required for the neuroprotective effect of CA. CA Reversed 6-OHDA Inhibition of GSTP Protein Expression in Lesioned Rats. The effects of CA on the expression of the GST family of proteins were further explored in rats with lesioning induced by 6-OHDA treatment in the right striatum. In the lesion group, the protein expression of GSTP was decreased in the striatum compared with that in the sham group. Conversely, pretreatment with CA reversed the

phosphorylation of IKKα/β and IκBα and the degradation of IκBα at 3 h (p < 0.05) (Figure 2B). The MAPK and PI3K/Akt signaling pathways are upstream mediators of NF-κB.22,23 Therefore, the activation of individual MAPK kinases and Akt at different time points was determined. These results showed that the phosphorylation of Akt was increased by CA up to 60 min (p < 0.05) (Figure 3). No difference in the phosphorylation of ERK, JNK, or p38 was detected during the time course. LY294002 Suppressed CA-Induced NF-κB Activation and GSTP Protein Expression. To address the role of the PI3K/Akt pathway in CA-induced NF-κB activation and GSTP protein expression, LY294002 (a PI3K/Akt inhibitor) was used. Immunoblotting indicated that pretreatment with LY294002 inhibited the CA-induced nuclear translocation of p65; the phosphorylation of Akt, IKKα/β, and IκBα; and the protein expression of GSTP (Figure 4A). The EMSA results showed that CA increased the GSTP-NF-κB DNA binding activity; however, pretreatment with LY294002 reduced this effect. The specificity of the DNA−protein interaction for NF-κB was demonstrated by a competitive assay with 200-fold of unlabeled D

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Figure 4. LY294002 inhibited CA-stimulated NF-κB activation and GSTP protein expression. SH-SY5Y cells were treated with 1 μM CA for 3 h in the presence or absence of 5 μM LY294002 (LY), which cells were exposed to for 1 h before CA treatment. (A) Protein expression was determined by Western blotting. β-Tubulin and PARP were used as the loading controls. The level in control cells was set at 1.0. One representative immunoblot out of three independent experiments is shown. Values are means ± SD, n = 3. Means without a common letter differ, p < 0.05. (B) Nuclear protein was prepared to determine NF-κB binding activity of human GSTP by EMSA assay. Unlabeled double-stranded NF-κB (200 ng) was used to confirm specific binding. One representative experiment out of three independent experiments is shown.

Figure 5. LY294002 reduced the neuroprotective effect of CA. SHSY5Y cells were treated with 1 μM CA for 18 h in the presence or absence of 5 μM LY294002 (LY) and were then cultured with 100 μM 6-OHDA for an additional 12 or 18 h. LY294002 was added to the cells for 1 h before CA treatment. (A) Protein expression was determined by Western blotting. β-Tubulin was used as the loading control. The ratio of cleaved caspase 3 and caspase 3 was calculated. The level in control cells was set at 1.0. One representative immunoblot out of three independent experiments is shown. (B) Cell viability was measured by MTT assay. The viability in control cells was set at 100%. Values are means ± SD, n = 3. Means without a common letter differ, p < 0.05.

activation, and cell death.33 The methanolic extract of Hibiscus asper leaves can protect the rat temporal lobe from 6-OHDAincreased lipid peroxidation and DNA fragmentation by enhancing the activities of superoxide dismutase, glutathione peroxidase, catalase, and total glutathione content.34 Recently, several studies have reported that CA has neuroprotection capacity. CA and its metabolite (CA12-methyl ether) present in the brain after rats fed the rosemary extracts.35 Satoh et al.’s research also indicated that CA is able to penetrate the bloodbrain barrier and protects the brain against middle cerebral artery ischemia/reperfusion by increasing the glutathione level in mice.26 Furthermore, we previously suggested that CA protects SH-SY5Y cells from 6-OHDA-elicited apoptosis via mediation of the protein expression of the γ-glutamylcysteine ligase catalytic subunit and modifier subunit.28 In the present study, we further suggest that the attenuation of 6-OHDAinduced apoptosis by CA is associated with the protein expression of GSTP through the PI3K/Akt/NF-κB pathway. 6-OHDA, a selective catecholaminergic neurotoxin, induces hydrogen peroxide and hydroxyl radicals via monoamine oxidase or auto-oxidation. GST is highly efficient in

reduction in GSTP protein induced by lesioning in the striatum. However, no effects on the protein expression of GSTA or GSTM were seen (Figure 7).

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DISCUSSION Oxidative stress has been implicated in the development of Parkinson’s disease.31 Oxidative stress refers to the imbalanced redox equilibrium between the production of free radicals and the ability of cells to defend against them. In both in vivo and in vitro models of Parkinson’s disease, 6-OHDA has been widely used to cause reactive oxygen species generation and to induce apoptosis signaling, which leads to cell death. One feasible way to prevent free-radical-caused cellular damage is to enhance oxidative defense capacity through the induction of antioxidant enzymes. For example, investigators have shown that sulforaphane can prevent 6-OHDA-induced cleavage of caspase 3 in SH-SY5Y cells by increasing the total GSH level and NAD(P)H:quinone oxidoreductase-1.32 In another study, the coffee diterpene kahweol was shown to induce heme oxygenase-1 to protect SH-SY5Y cells from 6-OHDAstimulated reactive oxygen species generation, caspase 3 E

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Figure 7. Effect of CA on the protein expression of GSTP in 6-OHDA unilaterally lesioned rats. The level of GST family protein in the striatum was determined by Western blotting. β-Tubulin was used as the loading control. The level in control cells was set at 1.0. One representative immunoblot out of six independent experiments is shown. Values are means ± SD of six independent experiments. Means without a common letter differ, p < 0.05.

Moreover, the induction of GSTP by CA attenuated 6-OHDAinduced apoptosis and cell death (Figure 5A and B). In another study, overexpression of GSTP protected SHSY5Y cells against leucine-rich repeat kinase 2 (LRRK2) mutant-induced apoptosis. However, knockdown of endogenous GSTP expression by use of short hairpin RNA exacerbated LRRK2 mutant-induced neurotoxicity.41 An animal study further indicated that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP in both the substantia nigra and the striatum than are wild-type mice.12 Moreover, the brain of GSTP knockout mice under MPTP treatment was more susceptible to ubiquitin-proteasome system impairment and oxidative stress than was the brain of wild-type mice.42 The present results showed that the protein expression of GSTP declined in the striatum of 6-OHDA-lesioned rats, but this suppression of GSTP protein was reversed in the presence of CA (Figure 7). However, when cells were treated with GSTP siRNA, the ability of CA to ameliorate the effects of 6-OHDA on GSTP protein and cell survival was reduced (Figure 6B and C). GSTP siRNA also reversed the ability of CA to suppress the ratio of cleaved caspase 3 and caspase 3 induced by 6OHDA treatment (Figure 6B). These results suggest that GSTP may play a crucial role in the action of CA against 6OHDA-induced neurotoxicity. GSTP protein participates in catalytic detoxification, chaperone function, reaction of protein S-glutathionylation, as well as regulation of nitric oxide and kinase signaling.43 Among the properties, JNK signaling is regulated by GSTP.44 The report indicated that GSTP protects against hydrogen peroxide-caused cell death by controlling JNK activity.45 Furthermore, GSTP suppressed JNK activation through interaction with the C terminus.46 Therefore, GSTP serves as a JNK inhibitor and inhibits JNK-activated apoptosis signaling. Interesting, our previous study also showed that CA inhibits 6-OHDA-induced apoptosis and JNK activation. It is possible that the neuroprotection of CA is related to the inhibition of JNK activity by GSTP. Future study in this field is necessary. It has also been shown that the expression of GSTP is regulated by NF-κB.14 Activation of NF-κB is known to be associated with increases in neuronal cell survival. Lee et al.47 reported that overexpression of NF-κB in PC12 cells inhibits dopamine-induced DNA fragmentation and PARP degradation. By contrast, knockdown of the p50 subunit of NF-κB increases cell damage resulting from stimulation by the excitotoxin

Figure 6. GSTP siRNA decreased the neuroprotective effect of CA. SH-SY5Y cells were transfected with 50 nM nontargeting control siRNA (si-control) or GSTP siRNA (si-GSTP) for 24 h. Cells were treated with 1 μM CA for 18 h and were then treated with 100 μM 6OHDA for an additional 12 or 18 h. GSTP protein (A) and cleaved caspase 3, caspase 3, and GSTP proteins (B) were determined by Western blotting. β-Tubulin was used as the loading control. The ratio of cleaved caspase 3 and caspase 3 was calculated. The level in control cells was set at 1.0. One representative immunoblot out of three independent experiments is shown. (C) Cell viability was measured by MTT assay. The viability in control cells was set at 100%. Values are means ± SD, n = 3. Means without a common letter differ, p < 0.05.

detoxification of the oxidized metabolites of catecholamines and provides protection by preventing degenerative cellular processes.36 For example, Shao et al.37 reported that lithium treatment inhibits hydrogen peroxide-elicited cell death and DNA fragmentation in primary cultured rat cerebral cortical cells via GSTM and GSTA. However, cells treated with the GST inhibitor ethacrynic acid showed a reduced neuroprotective effect of lithium. In mammals, GSTA, GSTM, and GSTP are expressed in the central nervous system.38,39 Among the GSTs, GSTP is constitutively and predominantly expressed in dopaminergic neurons of the substantia nigra.39,40 In the present study, we found that CA significantly induced the protein expression and enzyme activity of GSTP, but not that of GSTA and GSTM, in SH-SY5Y cells (Figure 1A and B). F

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kainate in the hippocampal pyramidal neurons of mice.20 The NF-κB pathway is regulated by IKK complex. Activation of the IKK complex results in the phosphorylation, polyubiquitination, and subsequent degradation of IκB through the 26S proteasome.21 In SH-SY5Y cells, the mu-opioid receptor regulates neuronal survival through phosphorylation of IKK and activation of NF-κB.48 Kim et al.’s study also indicated that metallothionein-III promotes the phosphorylation and degradation of IκB, stimulation of NF-κB from the cytosol to the nucleus, and subsequently the reduction of hydrogen peroxideinduced neuro-2a cell death.23 Furthermore, NF-κB inhibitor attenuates the neuroprotective effect of metallothionein-III. Our data showed that CA treatment markedly increased the phosphorylation of IKKα/β and IκBα as well as the degradation of IκBα at 3 h (Figure 2B). Furthermore, CA induced translocation of NF-κB nuclear protein from 3 to 6 h (Figure 2A) and increased the NF-κB nuclear protein−DNA binding activity of the GSTP gene (Figure 4B). Thus, the neuroprotective effect of CA is likely associated with the expression of GSTP through the NF-κB signaling pathway in SH-SY5Y cells. Several studies have suggested that the ERK, JNK, and p38 kinase pathways are associated with the NF-κB pathway.23,49,50 Manecka et al.50 suggested that pituitary adenylate cyclaseactivating polypeptide increases PC12 cell survival and neuritogenesis by activating NF-κB signaling and that the ERK pathway is involved in this activation. JNK induced the expression of antiapoptosis gene cIAP-2 is mediated by the coordinated actions of Jun D and NF- κB.51 Jiang et al.’s49 research also exerted that p38 signaling induces the mRNA level of Bcl-2 and Bcl-XL antiapoptosis genes, and subsequently protects retinal cells from ischemia injury through the NF-κB pathway. However, in the present study, ERK, JNK, and p38 were not affected by CA treatment (Figure 3). In addition to MAPKs, NF-κB is also regulated by the PI3K/Akt pathway. Kim et al.23 showed that metallothionein-III reduces hydrogenperoxide- or doxorubicin-induced neuron cell death by activating NF-κB through the PI3K/Akt pathway. Mo et al.52 showed that intravitreal administration of 17β-estradiol protects rat retinal neurons from light-induced caspase 3 cleavage by activating the NF-κB pathway through the PI3K/Akt pathway and that LY294002 (a PI3K/Akt inhibitor) reversed these effects. Consistent with these findings, our results indicated that CA protects neuronal cells against 6-OHDA-induced neurotoxicity through the induction of GSTP expression and activation of the PI3K/Akt/NF-κB pathway (Figure 5A and B). Using a specific inhibitor of the PI3K/Akt pathway, we found that LY294002 inhibited CA-induced activation of Akt, phosphorylation of IKKα/β and IκBα, degradation of IκBα, increase in NF-κB nuclear translocation, and induction of NFκB binding activity, which in turn down-regulated GSTP protein expression (Figure 4 A and B) and induced cell apoptosis in SH-SY5Y cells (Figure 5 A and B). This evidence indicates that the Akt signaling pathway is important in the activation of NF-κB-driven transcriptional activation of GSTP for neuroprotection by CA. In contrast, ERK, JNK, and p38 kinase are dispensable for the neuroprotective effect. In conclusion, CA prevents 6-OHDA-induced cell death through up-regulation of GSTP in SH-SY5Y cells. The PI3K/ Akt/NF-κB pathway is likely related to the neuroprotective effect of CA. Therefore, CA might be a promising candidate for the prevention of Parkinson’s disease.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1886-4-22053366, ext. 7521. Fax: +1886-4-22062891. Author Contributions

All authors contributed equally to this work. Funding

This work was supported by the National Science Council (NSC 100-2320-B-039-005 and NSC 101-2320-B-039-052MY2) Notes

The authors declare no competing financial interest.

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ABBREVIATIONS CA, carnosic acid; DMSO, dimethyl sulfoxide; EMSA, electromobility gel shift assay; ERK, extracellular signalregulated kinase; GST, glutathione S-transferase; GSTP, pi form of glutathione S-transferase; IKK, IκB kinase; JNK, c-Jun NH2-terminal kinase; LRRK2, leucine-rich repeat kinase 2; MAPK, mitogen-activated protein kinase; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor-kappa B; 6-OHDA, 6-hydroxydopamine; PI3K/Akt pathway, phosphatidylinositide 3-kinase/protein kinase pathway; siRNA, small interfering RNA

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REFERENCES

(1) Lotharius, J., and Brundin, P. (2002) Pathogenesis of Parkinson’s disease: dopamine, vesicles and alpha-synuclein. Nat. Rev. Neurosci. 3, 932−942. (2) Dauer, W., and Przedborski, S. (2003) Parkinson’s disease: mechanisms and models. Neuron 39, 889−909. (3) Schober, A. (2004) Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell Tissue Res. 318, 215− 224. (4) Lu, K. T., Ko, M. C., Chen, B. Y., Huang, J. C., Hsieh, C. W., Lee, M. C., Chiou, R. Y., Wung, B. S., Peng, C. H., and Yang, Y. L. (2008) Neuroprotective effects of resveratrol on MPTP-induced neuron loss mediated by free radical scavenging. J. Agric. Food Chem. 56, 6910− 6913. (5) Ng, C. F., Ko, C. H., Koon, C. M., Xian, J. W., Leung, P. C., Fung, K. P., Chan, H. Y., and Lau, C. B. (2013) The Aqueous Extract of Rhizome of Gastrodia elata Protected Drosophila and PC12 Cells against Beta-Amyloid-Induced Neurotoxicity. J. Evidence-Based Complementary Altern. Med. 2013, 516741. (6) Son, Y., Byun, S. J., and Pae, H. O. (2013) Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol, a natural analog and a metabolite of resveratrol, against glutamate-mediated oxidative injury in HT22 neuronal cells. Amino Acids 45, 393−401. (7) Dringen, R., and Hirrlinger, J. (2003) Glutathione pathways in the brain. Biol. Chem. 384, 505−516. (8) Hayes, J. D., Flanagan, J. U., and Jowsey, I. R. (2005) Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 51−88. (9) Castro-Caldas, M., Neves Carvalho, A., Peixeiro, I., Rodrigues, E., Lechner, M. C., and Gama, M. J. (2009) GSTpi expression in MPTPinduced dopaminergic neurodegeneration of C57BL/6 mouse midbrain and striatum. J. Mol. Neurosci. 38, 114−127. (10) Johnson, J. A., el Barbary, A., Kornguth, S. E., Brugge, J. F., and Siegel, F. L. (1993) Glutathione S-transferase isoenzymes in rat brain neurons and glia. J. Neurosci. 13, 2013−2023. (11) Shi, M., Bradner, J., Bammler, T. K., Eaton, D. L., Zhang, J., Ye, Z., Wilson, A. M., Montine, T. J., Pan, C., and Zhang, J. (2009) Identification of glutathione S-transferase pi as a protein involved in Parkinson disease progression. Am. J. Pathol. 175, 54−65.

G

dx.doi.org/10.1021/tx5003063 | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

(12) Castro-Caldas, M., Carvalho, A. N., Rodrigues, E., Henderson, C., Wolf, C. R., and Gama, M. J. (2012) Glutathione S-transferase pi mediates MPTP-induced c-Jun N-terminal kinase activation in the nigrostriatal pathway. Mol. Neurobiol. 45, 466−477. (13) Ishisaki, A., Hayashi, H., Suzuki, S., Ozawa, K., Mizukoshi, E., Miyakawa, K., Suzuki, M., and Imamura, T. (2001) Glutathione Stransferase Pi is a dopamine-inducible suppressor of dopamineinduced apoptosis in PC12 cells. J. Neurochem. 77, 1362−1371. (14) Morceau, F., Duvoix, A., Delhalle, S., Schnekenburger, M., Dicato, M., and Diederich, M. (2004) Regulation of glutathione Stransferase P1−1 gene expression by NF-kappaB in tumor necrosis factor alpha-treated K562 leukemia cells. Biochem. Pharmacol. 67, 1227−1238. (15) Xia, C., Hu, J., Ketterer, B., and Taylor, J. B. (1996) The organization of the human GSTP1−1 gene promoter and its response to retinoic acid and cellular redox status. Biochem. J. 313, 155−161. (16) Karin, M., and Lin, A. (2002) NF-kappaB at the crossroads of life and death. Nat. Immunol. 3, 221−227. (17) Bhakar, A. L., Tannis, L. L., Zeindler, C., Russo, M. P., Jobin, C., Park, D. S., MacPherson, S., and Barker, P. A. (2002) Constitutive nuclear factor-kappa B activity is required for central neuron survival. J. Neurosci. 22, 8466−8475. (18) Kaltschmidt, B., Widera, D., and Kaltschmidt, C. (2005) Signaling via NF-kappaB in the nervous system. Biochim. Biophys. Acta 1745, 287−299. (19) Maggirwar, S. B., Sarmiere, P. D., Dewhurst, S., and Freeman, R. S. (1998) Nerve growth factor-dependent activation of NF-kappaB contributes to survival of sympathetic neurons. J. Neurosci. 18, 10356− 10365. (20) Yu, Z., Zhou, D., Bruce-Keller, A. J., Kindy, M. S., and Mattson, M. P. (1999) Lack of the p50 subunit of nuclear factor-kappaB increases the vulnerability of hippocampal neurons to excitotoxic injury. J. Neurosci. 19, 8856−8865. (21) Hayden, M. S., and Ghosh, S. (2004) Signaling to NF-kappaB. Genes Dev. 18, 2195−2224. (22) Tuyt, L. M., Dokter, W. H., Birkenkamp, K., Koopmans, S. B., Lummen, C., Kruijer, W., and Vellenga, E. (1999) Extracellularregulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes. J. Immunol. 162, 4893−4902. (23) Kim, H. G., Hwang, Y. P., Han, E. H., Choi, C. Y., Yeo, C. Y., Kim, J. Y., Lee, K. Y., and Jeong, H. G. (2009) Metallothionein-III provides neuronal protection through activation of nuclear factorkappaB via the TrkA/phosphatidylinositol-3 kinase/Akt signaling pathway. Toxicol. Sci. 112, 435−449. (24) Posadas, S. J., Caz, V., Largo, C., De la Gandara, B., Matallanas, B., Reglero, G., and De Miguel, E. (2009) Protective effect of supercritical fluid rosemary extract, Rosmarinus officinalis, on antioxidants of major organs of aged rats. Exp. Gerontol. 44, 383−389. (25) Russo, A., Lombardo, L., Troncoso, N., Garbarino, J., and Cardile, V. (2009) Rosmarinus officinalis extract inhibits human melanoma cell growth. Nat. Prod. Commun. 4, 1707−1710. (26) Satoh, T., Kosaka, K., Itoh, K., Kobayashi, A., Yamamoto, M., Shimojo, Y., Kitajima, C., Cui, J., Kamins, J., Okamoto, S., Izumi, M., Shirasawa, T., and Lipton, S. A. (2008) Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J. Neurochem. 104, 1116−1131. (27) Park, J. A., Kim, S., Lee, S. Y., Kim, C. S., Kim do, K., Kim, S. J., and Chun, H. S. (2008) Beneficial effects of carnosic acid on dieldrininduced dopaminergic neuronal cell death. NeuroReport 19, 1301− 1304. (28) Chen, J. H., Ou, H. P., Lin, C. Y., Lin, F. J., Wu, C. R., Chang, S. W., and Tsai, C. W. (2012) Carnosic acid prevents 6-hydroxydopamine-induced cell death in SH-SY5Y cells via mediation of glutathione synthesis. Chem. Res. Toxicol. 25, 1893−1901. (29) Tsai, C. W., Liu, K. L., Lin, Y. R., and Kuo, W. C. (2014) The mechanisms of carnosic acid attenuates tumor necrosis factor-alpha-

mediated inflammation and insulin resistance in 3T3-L1 adipocytes. Mol. Nutr. Food Res. 58, 654−664. (30) Lii, C. K., Liu, K. L., Cheng, Y. P., Lin, A. H., Chen, H. W., and Tsai, C. W. (2010) Sulforaphane and alpha-lipoic acid upregulate the expression of the pi class of glutathione S-transferase through c-jun and Nrf2 activation. J. Nutr. 140, 885−892. (31) Wang, X., and Michaelis, E. K. (2010) Selective neuronal vulnerability to oxidative stress in the brain. Front. Aging Neurosci. 2, 1−13. (32) Tarozzi, A., Morroni, F., Merlicco, A., Hrelia, S., Angeloni, C., Cantelli-Forti, G., and Hrelia, P. (2009) Sulforaphane as an inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma cell line. J. Neurochem. 111, 1161− 1171. (33) Hwang, Y. P., and Jeong, H. G. (2008) The coffee diterpene kahweol induces heme oxygenase-1 via the PI3K and p38/Nrf2 pathway to protect human dopaminergic neurons from 6-hydroxydopamine-derived oxidative stress. FEBS Lett. 582, 2655−2662. (34) Hritcu, L., Foyet, H. S., Stefan, M., Mihasan, M., Asongalem, A. E., and Kamtchouing, P. (2011) Neuroprotective effect of the methanolic extract of Hibiscus asper leaves in 6-hydroxydopaminelesioned rat model of Parkinson’s disease. J. Ethnopharmacol. 137, 585−591. (35) Romo Vaquero, M., Garcia Villalba, R., Larrosa, M., YanezGascon, M. J., Fromentin, E., Flanagan, J., Roller, M., Tomas-Barberan, F. A., Espin, J. C., and Garcia-Conesa, M. T. (2013) Bioavailability of the major bioactive diterpenoids in a rosemary extract: metabolic profile in the intestine, liver, plasma, and brain of Zucker rats. Mol. Nutr. Food Res. 57, 1834−1846. (36) Baez, S., Segura-Aguilar, J., Widersten, M., Johansson, A. S., and Mannervik, B. (1997) Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes. Biochem. J. 324, 25−28. (37) Shao, L., Cui, J., Young, L. T., and Wang, J. F. (2008) The effect of mood stabilizer lithium on expression and activity of glutathione stransferase isoenzymes. Neuroscience 151, 518−524. (38) Beiswanger, C. M., Diegmann, M. H., Novak, R. F., Philbert, M. A., Graessle, T. L., Reuhl, K. R., and Lowndes, H. E. (1995) Developmental changes in the cellular distribution of glutathione and glutathione S-transferases in the murine nervous system. Neurotoxicology 16, 425−440. (39) Awasthi, Y. C., Sharma, R., and Singhal, S. S. (1994) Human glutathione S-transferases. Int. J. Biochem. 26, 295−308. (40) Castro-Caldas, M., Milagre, I., Rodrigues, E., and Gama, M. J. (2009) Glutathione S-transferase pi regulates UV-induced JNK signaling in SH-SY5Y neuroblastoma cells. Neurosci. Lett. 451, 241− 245. (41) Chen, J., Liou, A., Zhang, L., Weng, Z., Gao, Y., Cao, G., Zigmond, M. J., and Chen, J. (2012) GST P1, a novel downstream regulator of LRRK2, G2019S-induced neuronal cell death. Front. Biosci., Elite Ed. 4, 2365−2377. (42) Carvalho, A. N., Marques, C., Rodrigues, E., Henderson, C. J., Wolf, C. R., Pereira, P., and Gama, M. J. (2013) Ubiquitin-proteasome system impairment and MPTP-induced oxidative stress in the brain of C57BL/6 wild-type and GSTP knockout mice. Mol. Neurobiol. 47, 662−672. (43) Zhang, J., Grek, C., Ye, Z. W., Manevich, Y., Tew, K. D., and Townsend, D. M. (2014) Pleiotropic functions of glutathione Stransferase P. Adv. Cancer Res. 122, 143−175. (44) Adler, V., Yin, Z., Fuchs, S. Y., Benezra, M., Rosario, L., Tew, K. D., Pincus, M. R., Sardana, M., Henderson, C. J., Wolf, C. R., Davis, R. J., and Ronai, Z. (1999) Regulation of JNK signaling by GSTp. EMBO J. 18, 1321−1334. (45) Yin, Z., Ivanov, V. N., Habelhah, H., Tew, K., and Ronai, Z. (2000) Glutathione S-transferase p elicits protection against H2O2induced cell death via coordinated regulation of stress kinases. Cancer Res. 60, 4053−4057. H

dx.doi.org/10.1021/tx5003063 | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

(46) Wang, T., Arifoglu, P., Ronai, Z., and Tew, K. D. (2001) Glutathione S-transferase P1−1 (GSTP1−1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus. J. Biol. Chem. 276, 20999−21003. (47) Lee, H. J., Kim, S. H., Kim, K. W., Um, J. H., Lee, H. W., Chung, B. S., and Kang, C. D. (2001) Antiapoptotic role of NF-kappaB in the auto-oxidized dopamine-induced apoptosis of PC12 cells. J. Neurochem. 76, 602−609. (48) Liu, A. M., and Wong, Y. H. (2005) Mu-opioid receptormediated phosphorylation of IkappaB kinase in human neuroblastoma SH-SY5Y cells. Neurosignals 14, 136−142. (49) Jiang, S. Y., Zou, Y. Y., and Wang, J. T. (2012) p38 mitogenactivated protein kinase-induced nuclear factor kappa-light-chainenhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury. Mol. Vis. 18, 2096−2106. (50) Manecka, D. L., Mahmood, S. F., Grumolato, L., Lihrmann, I., and Anouar, Y. (2013) Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes both survival and neuritogenesis in PC12 cells through activation of nuclear factor kappaB (NF-kappaB) pathway: involvement of extracellular signal-regulated kinase (ERK), calcium, and c-REL. J. Biol. Chem. 288, 14936−14948. (51) Lamb, J. A., Ventura, J. J., Hess, P., Flavell, R. A., and Davis, R. J. (2003) JunD mediates survival signaling by the JNK signal transduction pathway. Mol. Cell 11, 1479−1489. (52) Mo, M. S., Li, H. B., Wang, B. Y., Wang, S. L., Zhu, Z. L., and Yu, X. R. (2013) PI3K/Akt and NF-kappaB activation following intravitreal administration of 17beta-estradiol: neuroprotection of the rat retina from light-induced apoptosis. Neuroscience 228, 1−12.

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