Molecular and Cellular Endocrinology 400 (2015) 21–31

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Review

Attenuation of mitochondrial and nuclear p38α signaling: A novel mechanism of estrogen neuroprotection in cerebral ischemia Dong Han a,1, Erin L. Scott b,1, Yan Dong b, Limor Raz b, Ruimin Wang b,c, Quanguang Zhang b,* a

Jiangsu Key Laboratory of Anesthesiology, Xuzhou Medical College, Jiangsu 221004, China Department of Neuroscience and Regenerative Medicine, Medical College of Georgia at Georgia Regents University, Augusta, GA 30912, USA c Neurobiology Institute of Medical Research Centre, Hebei United University, Tangshan, Hebei 06300, China b

A R T I C L E

I N F O

Article history: Received 19 October 2014 Received in revised form 16 November 2014 Accepted 17 November 2014 Available online 25 November 2014 Keywords: Global cerebral ischemia Estrogen Neuroprotection p38 Apoptosis

A B S T R A C T

P38 mitogen-activated protein kinase (MAPK) is a pro-apoptotic and pro-inflammatory protein that is activated in response to cellular stress. While p38 is known to be activated in response to cerebral ischemia, the precise role of p38 and its isoforms in ischemia-induced neuronal apoptosis remains unclear. In the current study, we examined the differential activation and functional roles of p38α and p38β MAPK isoforms in short-term ovariectomized female rats treated with either the neuroprotective ovarian hormone 17beta-estradiol (E2) or placebo in a model of global cerebral ischemia (GCI). GCI induced biphasic activation of total p38 in the hippocampal CA1, with peaks at 30 min and 1 day after 10-min ischemiareperfusion. Further study demonstrated that activated p38α, but not p38β, translocated to the nucleus 30 min and 3 h post reperfusion, and that this event coincided with increased phosphorylation of activating transcription factor 2 (ATF2), a p38 target protein. Intriguingly, activated p38α was also enhanced in mitochondrial fractions of CA1 neurons 1 day after GCI, and there was loss of mitochondrial membrane potential, as well as enhanced cytochrome c release and caspase-3 cleavage at 2 days post GCI. Importantly, E2 prevented the biphasic activation of p38, as well as both nuclear and mitochondrial translocation of p38α after GCI, and these findings correlated with attenuation of mitochondrial dysfunction and delayed neuronal cell death in the hippocampal CA1. Furthermore, administration of a p38 inhibitor was able to mimic the neuroprotective effects of E2 in the hippocampal CA1 region by preventing nuclear and mitochondrial translocation of p38α, loss of mitochondrial membrane potential, and neuronal apoptosis. As a whole, this study suggests that changes in subcellular localization of the activated p38α isoform are required for neuronal apoptosis following GCI, and that E2 exerts robust neuroprotection, in part, through dual inhibition of activation and subcellular trafficking of p38α. Published by Elsevier Ireland Ltd.

Contents 1. 2.

Introduction ........................................................................................................................................................................................................................................................... Materials and methods ...................................................................................................................................................................................................................................... 2.1. Animals and drug administration .................................................................................................................................................................................................... 2.2. Transient forebrain ischemia ............................................................................................................................................................................................................. 2.3. Tissue preparation, brain homogenates and subcellular fractionation ............................................................................................................................... 2.4. Immunoprecipitation and Western blotting ................................................................................................................................................................................ 2.5. Immunofluorescence staining ........................................................................................................................................................................................................... 2.6. Histological analysis ............................................................................................................................................................................................................................. 2.7. Kinase activity assay ............................................................................................................................................................................................................................. 2.8. Mitochondrial membrane potential (MMP) measurement .....................................................................................................................................................

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Grant Sponsor: AHA; Grant Number: 10SDG256009. * Corresponding author. Department of Neuroscience and Regenerative Medicine, Medical College of Georgia at Georgia Regents University, 1120 15th Street, CA-3020, Augusta, GA 30912, USA. Tel.: +1 706 721 8483; fax: +1 706 721 8685. E-mail address: [email protected] (Q. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.mce.2014.11.010 0303-7207/Published by Elsevier Ireland Ltd.

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2.9. Statistical analysis ................................................................................................................................................................................................................................. 24 Results ..................................................................................................................................................................................................................................................................... 24 3.1. Biphasic activation of p38 in hippocampal CA1 region following GCI and the inhibitory effects of E2 .................................................................. 24 3.2. Effects of E2 on GCI-induced p38α nuclear translocation and p38α activity in the vulnerable hippocampal CA1 region of ovariectomized rats .............................................................................................................................................................................................................................................................. 24 3.3. Effect of E2 on GCI-induced nuclear activation of ATF2 in the vulnerable hippocampal CA1 region at the early reperfusion stage ........... 25 3.4. Enhanced activity and mitochondrial translocation of p38α in the hippocampal CA1 at the late reperfusion stage after GCI and inhibition by E2 ........................................................................................................................................................................................................................................................... 26 3.5. Effects of E2 and p38 inhibitor (p38-I) on GCI-induced depolarization of mitochondrial membrane potential in hippocampal CA1 neurons of ovariectomized rats ......................................................................................................................................................................................................................... 26 3.6. Effects of E2 and p38 inhibitor (p38-I) on cytochrome c release, caspase-3 activation, and apoptosis in hippocampal CA1 neurons of ovariectomized rats ........................................................................................................................................................................................................................................ 26 3.7. Effects of p38 inhibitor on GCI-induced nuclear and mitochondrial translocation of p38 and on GCI-induced apoptosis in the hippocampal CA1 of ovariectomized rats ................................................................................................................................................................................................................ 28 Discussion .............................................................................................................................................................................................................................................................. 28 Acknowledgements ............................................................................................................................................................................................................................................. 30 Appendix: Supplementary material .............................................................................................................................................................................................................. 30 References .............................................................................................................................................................................................................................................................. 30

1. Introduction P38 MAPK belongs to the stress-activated serine/threonine protein kinases that are responsive to mitogenic stimuli and cellular stress (Han et al., 1994; Obata et al., 2000). Studies indicate that p38 MAPK is activated by dual phosphorylation at Thr180 and Tyr182 in response to a wide range of stimuli (Raingeaud et al., 1995). Once activated, p38 MAPK translocates to the nucleus and subsequently phosphorylates and activates multiple downstream transcription factors, including ATF2, p53 and MEF2C, thereby controlling stressresponsive gene expression (Ben-Levy et al., 1998; Cardaci et al., 2010; de Nadal et al., 2011; Wood et al., 2009; Zhu et al., 2002). It is well known that activation of p38 MAPK plays a pivotal role in apoptotic cell death, as inhibition of p38 MAPK activation attenuates the mitochondrial apoptosis pathway (Barone et al., 2001; Ghatan et al., 2000; Gomez-Lazaro et al., 2007, 2008; Kim et al., 2004, 2006; Liu et al., 2009; Shou et al., 2003; Yang et al., 2013). On the other hand, activation of p38 MAPK in the hippocampus has also been implicated in the development of neuronal tolerance to seizures and ischemia (Bi et al., 2014; Dreixler et al., 2009; Jiang et al., 2005; Nishimura et al., 2003; Sun et al., 2010). 17β-Estradiol (E2) is an endogenous steroid hormone that plays an important role in the regulation of neurotrophism and neuronal survival, and E2 is thought to be protective against various neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and stroke (Brann et al., 2007, 2012). While much is known about the physiological role of E2 signaling, the molecular mechanisms underlying E2’s neuroprotective effects in cerebral ischemia have not been fully characterized. Similar to other MAP kinases, such as the extracellular signal-related kinases (ERKs) and the c-Jun N-terminal kinases (JNKs), p38 MAPK is highly expressed in the mammalian brain and is activated following both global and focal cerebral ischemia (Ferrer et al., 2003; Lennmyr et al., 2002; Roy Choudhury et al., 2014; Sugino et al., 2000; Zhu et al., 2014). Intriguingly, E2 was shown to inhibit the pro-apoptotic JNK pathway activation (Wang et al., 2006; Zhang et al., 2008) and increase anti-apoptotic ERKs signaling (Jover-Mengual et al., 2007; Yang et al., 2010) following cerebral ischemic injury, thereby promoting neuronal survival. However, it is unknown if activation of p38 MAPK is implicated in neuronal damage following global cerebral ischemia (GCI) in ovariectomized rats. In addition, it is unclear whether neural p38 MAPK activation can be regulated by systemic E2 replacement and whether this regulation contributes to the neuroprotective effect of E2. To address these questions, we utilized a well-established model of GCI and characterized both neuronal p38 MAPK activation and its regulation by E2 in the

hippocampus of ovariectomized rats. Both p38α and p38β isoforms were studied, since these two isoforms are predominately expressed in mature neurons of the rodent hippocampus (Lee et al., 2000). 2. Materials and methods 2.1. Animals and drug administration All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize the number of animals used and to reduce animal suffering and distress. Adult (3-month old) female Sprague–Dawley rats (Harlan) were used for the study. Animals were housed in a temperature-controlled facility on a 12-h light/dark cycle with ad libitum access to phytoestrogen-free chow diet. To exclude the effect of endogenous ovarian steroids, all rats were bilaterally ovariectomized under isoflurane anesthesia. At the time of ovariectomy (OVX), 17β-estradiol (E2, 0.017 mg) or placebo (Pla, 20% β-cyclodextrin) osmotic mini-pumps (14-day release, model 1002, Alzet, Durect Corp., USA) were implanted subcutaneously in the upper mid-back region. This dose of E2 was used to mimic low, physiological E2 levels produced during Diestrus I (10–15 pg/mL serum E2) (Zhang et al., 2008). In a subset of studies, 10 μL of SB203580 (100 μM in 1% DMSO, Tocris Bioscience, Ellisville, MO), a specific inhibitor of P38 MAPK, was administrated by intracerebroventricular (i.c.v.) infusion 20 min before ischemia based on the following stereotaxic coordinates from Bregma: anterior/ posterior −0.8 mm, medial/lateral ±1.5 mm, dorsal/ventral −3.5 mm. For i.c.v. injections, anesthetized rats were placed on a stereotaxic instrument. Drug infusion was performed using a Hamilton microsyringe at a flow rate of 1 μL/min, and the needle was left in situ for 5 min before the complete 2 min retraction. 2.2. Transient forebrain ischemia To induce transient forebrain ischemia, all animals (except sham control) underwent global cerebral ischemia (GCI) via four-vessel occlusion 7 days after OVX, as previously described (Zhang et al., 2011). Briefly, under chloral hydrate (350 mg/kg, i.p.) anesthesia on day 6 post OVX, the first and second cervical vertebral bodies were exposed through a midline incision, and the vertebral arteries were permanently occluded via electrocauterization. Then, both common carotid arteries (CCAs) were isolated and loosely ligated with surgical sutures without interrupting blood flow, and the incision was closed with tissue glue. Animals were allowed to recover for 24 h, and then, on day 7 post OVX, transient GCI was induced

D. Han et al./Molecular and Cellular Endocrinology 400 (2015) 21–31

by occluding the CCAs for 10 minutes with hemostatic clips. Restoration of arterial blood flow was confirmed before the wound was closed. Subjects were monitored for bilateral pupil dilation, loss of righting reflex, and unresponsiveness to light to ensure successful forebrain ischemia. Rectal temperature was maintained at 37 ± 0.5 °C via an infrared lamp and a thermal blanket coupled to a rectal thermistor. Sham-operated animals underwent identical surgical procedures except for carotid artery occlusion.

2.3. Tissue preparation, brain homogenates and subcellular fractionation To collect fresh brain tissue, rats were sacrificed by decapitation under deep anesthesia, and the brains were rapidly removed. The hippocampal CA1 regions were quickly separated along the hippocampal fissure on ice using a standardized microdissection procedure. The separated tissues were homogenized as previously described (Shi et al., 2014), using a motor-driven Teflon homogenizer in ice-cold homogenization buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 12 mM β-glycerophosphate, 1% Triton X-100) plus inhibitors of proteases and enzymes (Thermo Scientific, Rockford, IL). The homogenates were vigorously mixed for 20 min on a rotator and centrifuged at 15,000 × g for 30 min at 4 °C to obtain total protein fractions. For subcellular fractionation, tissues were homogenized in ice-cold buffer A containing 10 mM HEPES (pH 7.9), 12 mM β-glycerophosphate, and inhibitors of proteases and enzymes. After the addition of NP-40 to 0.6%, the homogenates were allowed to sit on ice for 10 min and vigorously vortexed for 30 s before centrifugation at 800 × g for 10 min. The resulting supernatants were centrifuged at 17,000 × g for 20 min at 4 °C to yield crude mitochondrial fractions in the pellets and cytosolic fractions in the supernatants. Nuclear pellets were dissolved with buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 20% glycerine), rocked at 4 °C for 30 min, and centrifuged at 12,000 × g for 20 min to yield the nuclear fractions. Protein concentrations were determined via Modified Lowry Protein Assay (Pierce, Rockford, IL).

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was calculated for each group for statistical comparison, and data were expressed as fold changes versus sham control group for graphical representation.

2.5. Immunofluorescence staining Immunofluorescence staining was performed as previously described (Zhang et al., 2009). Briefly, animals were deeply anesthetized with isoflurane and perfused transcardially with 0.1 M phosphatebuffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4). Brains were removed, postfixed in the same fixative overnight, and cryoprotected with 30% sucrose in 0.1 M PB at 4 °C until they sank. The tissues were then embedded in optimal cutting temperature (OCT) compound and 20μm-thick frozen sections were cut through the coronal plane of the dorsal hippocampus. Sections containing intact hippocampus were collected and utilized for single/double immunofluorescence staining as previously described (Zhang et al., 2009). Briefly, coronal brain sections were blocked with 10% normal donkey serum for 1 h at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with appropriate primary antibodies in PBS supplemented with 0.1% Triton X-100 overnight at 4 °C. After primary antibody incubation, sections were washed 4 × 10 min at room temperature, followed by incubation with the appropriate Alexa Fluor 568/488 donkey anti-rabbit/mouse secondary antibody (1:500; Invitrogen Corporation, Carlsbad, CA) for 1 h at room temperature. Sections were then washed with PBS and briefly with water and mounted with water-based mounting medium containing antifading agents (Biomeda, Fisher Scientific, Pittsburgh, PA). Confocal images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss, Germany) using 40× oil immersion Neofluor objective (NA, 1.3) with the image size set at 1024 × 1024 pixels. The captured images were viewed and analyzed using LSM510 Meta imaging software.

2.6. Histological analysis 2.4. Immunoprecipitation and Western blotting For immunoprecipitation (IP), a previously described standard protocol (Zhang et al., 2009) was used with minor modifications. Samples containing 400 μg of protein were diluted fourfold in HEPES buffer without detergent. Incubation with the anti-p38α or antip38β antibody (5 μg each, Santa Cruz Biotechnology, Inc.) was performed overnight at 4 °C. Immunocomplexes were captured by incubation with 20 μL protein A/G sepharose beads at 4 °C for an additional 2 h and subsequent washing three times with ice-cold HEPES buffer. For Western blot analysis, samples were mixed with Laemmli loading buffer and boiled for 5 min. Protein samples (30 μg each) were separated by 4–20% SDS–PAGE. Proteins were transferred to a PVDF immobilon-FL membrane (Millipore), blocked in Odyssey Blocking Buffer for 1 h, and incubated with primary antibody at 4 °C overnight. Antibodies against phosphorylated p38 (pp38, Thr180/Tyr182), total p38, cytochrome c (Cyt c), β-actin and COX4 were from Santa Cruz Biotechnology, Inc. Phosphorylated ATF2 (p-ATF2, Thr69/71) and cleaved caspase-3 antibodies were from Cell Signaling Technology, Inc. NeuN antibody was from EMD Millipore, Inc. The membrane was washed with PBS containing 0.1% Tween 20 to remove unbound antibody, followed by incubation with secondary Alexa Fluor 680 goat anti-rabbit/mouse antibody for 1 h at room temperature. Blots were visualized using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE), and semi-quantitative analysis of fluorescent signals was performed using ImageJ analysis software (National Institutes of Health, Bethesda, MD). A mean ± SE

Histological examination was performed on free-floating coronal hippocampal sections by NeuN and TUNEL staining as described previously by our lab (Zhang et al., 2009). Briefly, 7 days after cerebral ischemia or sham surgery, animals were deeply anesthetized, and brain sections were prepared as mentioned earlier. Sections were stained with an antibody against NeuN (1:500, Millipore Bioscience). Images were then captured on an LSM510 Meta confocal laser microscope. NeuN-positive cells with intact and round nuclei appearing in the hippocampal CA1 pyramidal layer were counted as surviving neurons. TUNEL staining was performed on free-floating coronal sections using an In Situ Cell Death Detection Kit (Roche, Penzberg, Germany), according to manufacturer’s instructions. Briefly, after permeabilization with 10 μg/mL proteinase K in 10 mM Tris/ HCl (pH 7.4) for 15 min and blocking for 1 h, slides were incubated with TUNEL reaction mixture including enzyme solution (TdT) and tetramethylrhodamine (TMR)-labeled TUNEL-positive nucleotides in a humidified chamber for another 1 h at 37 °C. Slides for negative control were incubated with the label solution without terminal transferase for TUNEL. Images were acquired on a LSM510 Meta confocal microscope. For quantitative assessment, the number of surviving neurons and the number of TUNEL-positive cells per 250 μm length of medial CA1 pyramidal cell layer was counted bilaterally in 3–5 representative sections (200 μm apart, approximately 1.5–3.3 mm posterior to Bregma) per animal. Cell counts were then averaged to provide a single value for each animal. A mean ± SE was calculated from the data in each group, and statistical analysis was performed as will be described later.

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2.7. Kinase activity assay The activity of p38α or p38β MAPK was performed by measuring phosphorylated ATF2 (p-ATF2) levels in an in vitro kinase assay by using a p38 MAPK Assay Kit (#9820, Cell Signaling Technology. Inc.), according to manufacturer’s instructions. Briefly, p38α or p38β protein was immunoprecipitated as indicated earlier from nuclear protein samples or mitochondrial protein samples. Kinase reaction was performed in the presence of 100 μM cold ATP and 1 μg ATF-2 fusion protein for 30 min at 30 °C. The reaction was terminated by addition of 25 μL 3X SDS Sample Buffer. Phosphorylated ATF2 was then measured by Western blotting analysis using anti-phospho-ATF2 (Thr71) antibody. 2.8. Mitochondrial membrane potential (MMP) measurement Measurement of MMP was conducted using JC1 staining as described previously (Zhou et al., 2011). Briefly, JC1 (1 μL in 9 μL saline, #10009172, Cayman Chemical, Ann Arbor, MI) was bilaterally infused into the lateral ventricles 1 h prior to sacrifice. Animals were deeply anesthetized, and 20-μm-thick coronal hippocampal sections were prepared as mentioned earlier. Sections were washed with PBS and briefly with water, and sections were then mounted with DAPI VECTASHIELD solution (Vector Laboratories Inc.). JC1 fluorescence was captured on an LSM510 Meta confocal laser microscope using 40× oil immersion Neofluor objective (Ex/Em: 590/610 nm for JC1 aggregates, Ex/Em: 485/535 nm for JC1 monomers). JC1 fluorescent intensity was quantified using ImageJ analysis software and expressed by the red-to-green ratio of JC1 aggregates to monomers, with the control red-to-green ratio normalized to 1.0. The normalized JC-1 fluorescence ratio was compared between groups. 2.9. Statistical analysis Statistical analysis was performed using SigmaStat 3.5 software (SPSS, Inc., Chicago, IL). All values were expressed as mean ± SE. Results were obtained using one-way analysis of variance (ANOVA), followed by either Student–Newman–Keuls (for all pairwise group comparisons) or Dunnett’s (for multiple comparisons versus a control) post-hoc tests. When only two groups were compared, a Student’s t-test was used. Probability values p < 0.05 were considered to be statistically significant. 3. Results 3.1. Biphasic activation of p38 in hippocampal CA1 region following GCI and the inhibitory effects of E2 We first examined the temporal profile of total p38 protein expression and p38 MAPK activation (phosphorylation) in hippocampal CA1 region following GCI in adult ovariectomized rats with either Pla or E2 treatment. Fig. 1A and B shows Western blot results for levels of total p38 and phospho-p38 MAPKThr180/Tyr182 (p-p38) in the hippocampal CA1 region of sham and Pla (ischemic) animals. Dual phosphorylation of p38 MAPK at Thr180 and Tyr182 is known to activate p38. As shown in Fig. 1A and B, GCI induced a biphasic activation pattern of p38 MAPK in the hippocampal CA1 region – with an initial peak of p-p38 observed at 30 min after GCI, followed by a fall in p-p38 levels at 3 h and 6 h (to levels that were still statistically higher than the sham control) – and a second peak of p-p38 at 24–48 h after GCI. The GCI-induced elevation of p38 MAPK activation was specific for the CA1 region, as it was not observed in the more ischemia-resistant hippocampal CA3/DG regions (data not shown). Interestingly, p38 activation in the hippocampal CA1 was significantly reduced by E2 treatment at all time-points

examined following GCI (as compared to Pla). It should be noted that the change in p38 activation after GCI and its regulation by E2 was not due to a change in p38 expression, as total protein expression of p38 MAPK was not significantly changed following GCI in any group. In order to further characterize the p38 isoform(s) involved in activation following GCI, as well as examine the potential regulatory effects of E2, p38α and p38β proteins were immunoprecipitated from hippocampal CA1 homogenate at the early 30 min activation peak, and the immunoprecipitates were used for blotting of p-p38. As shown in Fig. 1C, Western blotting indicated that p38 phosphorylation was significantly elevated only in p38α immunoprecipitates of the Pla group 30 min after reperfusion. Furthermore, E2 treatment diminished this effect. Intriguingly, the phosphorylation level of the p38β isoform was unchanged after GCI, regardless of treatment. In addition, as shown in Fig. 1D, confocal immunofluorescence analysis for p38α/β (green), p-p38 (red) and merged images demonstrated that activated p-p38 strongly co-localized with the p38α, but not the p38β, isoform in the nucleus of hippocampal CA1 neurons. Thus, these results suggest that the p38α subtype is the main isoform activated in the CA1 region following GCI, and that E2 can prevent p38α activation in the ischemia-vulnerable hippocampal CA1 neurons in ovariectomized rats.

3.2. Effects of E2 on GCI-induced p38α nuclear translocation and p38α activity in the vulnerable hippocampal CA1 region of ovariectomized rats We next examined the subcellular distribution of p38α protein using Western blot analysis and immunohistochemistry. As shown in Fig. 2A, Western blotting indicated that p38α protein levels were significantly decreased in the cytoplasmic fractions of hippocampal CA1 proteins and simultaneously elevated in the nuclear protein fractions at the early reperfusion stage (30 min to 3 h) following GCI, compared to sham-operated controls. This simultaneous cytoplasmic loss and nuclear accumulation of p38α suggest that p38α quickly translocates to the nucleus following GCI. Of note, p38α nuclear translocation in hippocampal CA1 regions following GCI was inhibited in the E2-treated groups at the same time points, compared to placebo. In addition, confocal immunofluorescence staining showed that p38α immunoreactivity highly co-localized with NeuN (a neuronal nuclear marker) in Pla-treated animals at 3 h following GCI compared with sham control (Fig. 2B), providing further evidence of p38α nuclear translocation at 3 h following ischemic reperfusion. In contrast, co-localization of p38α and NeuN was diminished in hippocampal CA1 neurons of E2-treated rats, further supporting an inhibitory effect of E2 on p38α nuclear translocation following GCI. We also evaluated p38α enzymatic activity by measuring phosphorylated levels of the p38 target transcription factor ATF2 (phospho-ATF2, p-ATF2) with an in vitro kinase assay, using immunoprecipitated p38α from nuclear protein samples. Fig. 2C shows that p38α nuclear activity was elevated at early reperfusion time points (30 min and 3 h) in the placebo group, which was reduced to sham levels by E2 replacement. However, neither p38α nuclear translocation nor p38α activity was significantly changed at the relatively late reperfusion stage (2–48 h) following GCI in the Pla- or E2-treated groups, compared to sham control. Importantly, the nuclear translocation and kinase activity of the p38β isoform were also examined after GCI (Supplementary Fig. S1A and C), but no changes were observed in any group at the time points examined (sham, 30 min, 3 h, 6 h, and 24 h). Altogether, these data indicate that exogenous E2 replacement may effectively attenuate GCIinduced nuclear translocation and activation of p38α in vulnerable hippocampal CA1 neurons.

D. Han et al./Molecular and Cellular Endocrinology 400 (2015) 21–31

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Fig. 1. Effects of E2 on biphasic activation (phosphorylation) of p38 (p-p38) in the hippocampal CA1 region following GCI. (A, B) Western blot analyses of dually phosphorylated p38 (Thr180/Tyr182), total p38, and β-actin were performed with hippocampal CA1 total protein samples in the indicated groups of ovariectomized rats with placebo (Pla, −) or E2 (+) treatment. Levels of p38 activation (p-p38/total p38) were expressed as fold changes versus Pla group and compared to a sham ischemia group (Sham). (C, D) P38 activation mainly occurs via the p38α subunit. Hippocampal CA1 protein samples at reperfusion (R) 30 min were subjected to immunoprecipitation (IP) with antip38α or p38β antibodies, separately, and blotted with anti-p-p38 antibody. Note that p-p38 level only changes in p38α immunoprecipitates. Confocal analysis for p38α/ p38β (green), p-p38 (red) and merged images shows marked co-localization between the p38α subunit and p-p38 in nucleus of hippocampal CA1 neurons (magnification: 40×, scale bar = 50 μm). Data represent mean ± SE (n = 4). *P < 0.05 compared to sham. #P < 0.05 versus Pla group at the same time point. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.3. Effect of E2 on GCI-induced nuclear activation of ATF2 in the vulnerable hippocampal CA1 region at the early reperfusion stage

examined whether ischemic reperfusion promotes the activation (phosphorylation) of the downstream transcription factor ATF2 in hippocampal CA1 neurons and whether this change could be regulated by E2. Western blot analysis was performed on nuclear protein samples from the hippocampal CA1 region of ovariectomized rats

The increased accumulation of p38α in the neuronal nucleus may enhance p38 regulation of transcriptional activity. Therefore, we next

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Fig. 2. E2 prevents p38α nuclear translocation and activity at 30 min and 3 h after GCI. (A) Western blot analysis of p38α protein expression using nuclear (N) and cytoplasmic (C) samples from CA1 region at the indicated time points after GCI. β-actin and NeuN were used as loading controls for protein in the cytosol and nucleus, respectively. (B) Confocal images with NeuN (red) and p38α (green) staining show nuclear translocation of p38α at 3 h after GCI in Pla group, which was apparently attenuated by E2. Magnification: 40×, scale bar: 50 μm. (C) P38α enzymatic activity was determined by measuring phosphorylation of ATF2 (p-ATF2) in an in vitro kinase assay using immunoprecipitated p38α from nuclear protein samples. Note that p38α nuclear activity was only elevated at early reperfusion time points (30 min and 3 h), which was lowered to sham levels by E2. Data represent mean ± SE (n = 4–5). *P < 0.05 versus sham control, #P < 0.05 versus Pla group at the same time point.

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D. Han et al./Molecular and Cellular Endocrinology 400 (2015) 21–31

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Fig. 3. E2 prevents the nuclear activation of transcription factor ATF2 at 30 min and 3 h after GCI. (A, B) Nuclear protein samples from CA1 region with Pla or E2 treatment were subjected to Western blot analysis for p-ATF2 expression. (C) Confocal images with NeuN (red) and p-ATF2 (green) staining show nuclear activation (phosphorylation) of ATF2 at 3 h in Pla group, which was prevented by E2. Magnification: 40×, scale bar: 50 μm. *P < 0.05 versus sham control, #P < 0.05 versus Pla group at the same time point.

with Pla or E2 replacement. Of significant interest, as shown in Fig. 3A and B, results revealed that ATF2 was activated at 30 min to 3 h after ischemia, and this event was prevented by E2 treatment. In contrast, ATF2 activation status was not significantly changed at the late reperfusion stage (24–48 h) following GCI in the Pla- or E2-treated groups, compared with the sham control. Furthermore, the Western blot results were confirmed by double confocal microscopy analysis for p-ATF2 and NeuN. As shown in Fig. 3C, immunofluorescence staining showed that strong p-ATF2 immunoreactivity was detected in the nucleus of CA1 neurons at 3 h ischemic reperfusion in Pla-treated ovariectomized rats. However, only negligible p-ATF2 immunoreactivity was observed in the nucleus of sham-operated animals and E2-teated animals. These observations demonstrate that E2 can inhibit GCI-induced nuclear activation of the transcription factor ATF2 at the early reperfusion stage (30 min and 3 h), which is consistent with its inhibitory effect on p38α activation.

compared with sham control (Supplementary S1D). Importantly, both the mitochondrial translocation of p38α and the increase in p38α kinase activity were reduced in E2-treated animals. Furthermore, confocal immunofluorescence for the Mit marker COX4 (red) and p38α (green) demonstrated a high co-localization (yellow, white arrows) in the Platreated rats at 24 h after ischemic reperfusion (Fig. 4C). However, very little co-localization between COX4 and p38α was observed in the sham control and E2-treated animals. As a whole, these data demonstrate that p38 MAPK activation at the late ischemic reperfusion stage involves the targeting of p38α to the mitochondria and that E2 has an inhibitory effect on the activation of p38α following GCI in the ischemia-vulnerable CA1 region.

3.4. Enhanced activity and mitochondrial translocation of p38α in the hippocampal CA1 at the late reperfusion stage after GCI and inhibition by E2

Mitochondrial membrane potential (MMP) was further evaluated under confocal microscopy in the hippocampal CA1 neurons of ovariectomized rats by using the fluorescence changes of the voltagedependent dye JC-1 at 1 d after ischemic reperfusion. In healthy mitochondria, JC-1 shifts from monomeric form (green) to the aggregate form (red) (Fig. 5A, sham group). JC-1 fluorescence was normalized as the control red-to-green ratio, which was converted to 1.0 in the sham control group. As shown in Fig. 5, in Pla- and Veh-treated groups, the normalized JC-1 fluorescence in neuronal mitochondria after 1 d reperfusion was markedly decreased in the cytoplasm of CA1 pyramidal neurons, suggesting a GCI-induced mitochondrial depolarization and potential collapse of MMP. Intriguingly, treatment with either E2 or the specific p38 inhibitor SB203580 (p38-I) reversed the GCI-induced change in JC-1 equilibrium, compared with Pla- and Veh-treated controls, indicating a preservation of MMP and the presence of healthy mitochondria. Taken as a whole, these results suggest that E2 is able to prevent mitochondrial impairment from ischemic insult, at least partly, via down-regulating p38 MAPK activation.

Since we noted that GCI induced a biphasic activation of p38 in hippocampal CA1 neurons, we next aimed to determine the potential role of p38 activation at the later reperfusion stage (24–48 h after GCI). To accomplish this aim, Western blot analysis was used to examine the temporal profile of p38α protein expression in mitochondrial (Mit) fraction samples from the rat hippocampal CA1 region after GCI. As shown in Fig. 4A, p38α expression level in mitochondrial fractions was not significantly changed at 30 min, 3 h, or 6 h after GCI in ovariectomized rats that received Pla treatment, as compared to sham control. However, mitochondrial accumulation of p38α was markedly increased 24– 48 h after GCI compared with sham control. Importantly, COX4, a marker for Mit fractions, was strongly expressed in the Mit fraction samples, suggesting enrichment of Mit protein. Weak immunoreactivity of p38β expression was also detected in the Mit fractions, with no significant changes in any time point examined (Supplementary S1B). We further evaluated whether the Mit accumulation of p38α led to an increase in p38α activity. P38α and p38β activity was determined by measuring p-ATF2 levels in an in vitro kinase assay using immunoprecipitated p38α or p38β from Mit protein samples, respectively. As shown in Fig. 4B, p38α activity in Mit fractions was elevated at the late stage (24–48 h) following ischemic reperfusion in the Pla-treated animals, compared to sham control. Consistent with weak p38β immunoreactivity in the Mit fractions, we detected no significant changes of p38β activity

3.5. Effects of E2 and p38 inhibitor (p38-I) on GCI-induced depolarization of mitochondrial membrane potential in hippocampal CA1 neurons of ovariectomized rats

3.6. Effects of E2 and p38 inhibitor (p38-I) on cytochrome c release, caspase-3 activation, and apoptosis in hippocampal CA1 neurons of ovariectomized rats To determine the possible downstream effects following ischemic reperfusion-induced mitochondrial translocation of p38α and MMP collapse in CA1 neurons, cytoplasmic protein samples (without the mitochondrial portion) from the CA1 region at 48 h after GCI

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were examined by Western blotting. As indicated in Fig. 6A, cytoplasmic expression of Cyt c and cleaved (activated) caspase-3 was elevated in Pla- and Veh-treated animals at 48 h after GCI. As expected, Western blot analysis showed that cytosolic Cyt c expression was significantly decreased in both E2 and p38 inhibitortreated animals, compared with Pla or Veh controls, respectively.

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Fig. 6. E2 and p38 inhibitor attenuate GCI-induced mitochondrial cytochrome c release, caspase-3 activation, and neuronal apoptosis. (A) Cytoplasmic protein samples from hippocampal CA1 at 48 h after GCI were subjected to Western blot analysis for cytochrome c (cyt c) and cleaved (activated) caspase-3. (B) Confocal images show neuronal activation of caspase-3 at 48 h after GCI in Pla group, which was prevented by treatment with E2 or p38 inhibitor (p38-I). (C) Both E2 and p38 inhibitor attenuated the number of TUNEL-positive apoptotic cells in hippocampal CA1 region at 5 d after GCI. Magnification: 40×, scale bar: 50 μm. Data represent mean ± SE (n = 4 in A and B, n = 6–7 in C). *P < 0.05 versus sham group, #P < 0.05 versus Pla or Veh control group.

hippocampal CA1 neurons of E2 or p38 inhibitor-treated animals. These data suggest that p38α translocation to the mitochondria observed following GCI is associated with subsequent release of Cyt c from mitochondria into the cytosol and caspase-3 activation in hippocampal CA1 neurons. In order to analyze GCI-induced apoptotic cell death of hippocampal CA1 neurons, coronal brain sections were subjected to TUNEL staining 5 d following ischemic reperfusion. As shown in Fig. 6C, most of the hippocampal CA1 neurons were positive for TUNEL staining 5 d after ischemia in Pla-treated animals. Importantly, no TUNEL-positive cells were observed in the sham control. Quantitative analyses indicated that the number of TUNELpositive cells was significantly decreased in both the E2 and p38 inhibitor-treated animals, compared to ischemic Pla-control. As such, these data indicate the ability of E2 to inhibit the intrinsic apoptotic pathway is associated with the attenuation of p38 MAPK signaling. 3.7. Effects of p38 inhibitor on GCI-induced nuclear and mitochondrial translocation of p38 and on GCI-induced apoptosis in the hippocampal CA1 of ovariectomized rats To investigate whether the nuclear translocation of p38α (at the early stage after GCI) and mitochondrial translocation of p38α (at

the late stage after GCI) were associated with GCI-induced delayed neuronal cell death in the hippocampal CA1 region, the specific p38 inhibitor SB203580 was given via intracerebroventricular injection 20 minutes prior to ischemia. Western blot analysis was carried out for p38α protein expression in nuclear protein samples at 3 h reperfusion and in mitochondrial protein samples at 1 d reperfusion. As seen in Fig. 7A and B, the results revealed that both the nuclear and mitochondrial translocations of p38α were prevented by the p38 inhibitor, compared with Pla and Veh control groups. Furthermore, histological evaluation of cell survival, as shown in Fig. 7C, revealed that pretreatment with the p38 inhibitor resulted in robust neuroprotection of the rat hippocampal CA1 region from GCI, as evidenced by a significantly increased number of surviving neurons in the medial CA1 region. These data suggest that both E2 and the p38 inhibitor SB203580 protect against GCIinduced delayed neuronal cell death, in part, through inhibiting p38α translocation to the nucleus and mitochondria. 4. Discussion The current study provides novel information on p38 activation, regulation, and role in the hippocampal CA1 region after GCI. P38 activation was shown to exhibit a biphasic activation pattern

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Fig. 7. P38 inhibitor attenuates nuclear and mitochondrial translocation of p38α and protects neurons in the hippocampal CA1. (A, B) Western blot analysis for p38α expression in nuclear and mitochondrial protein samples from CA1 region at 3 h and 1 d after GCI, respectively. Note that both p38α nuclear translocation and mitochondrial translocation of were prevented by p38 inhibitor. (C) Pretreatment with p38 inhibitor resulted in significant neuroprotection from GCI, as evidenced by an increased number of surviving neurons in the medial hippocampal CA1 region. NeuN (red), Fluoro-Jade B (green). Data represent mean ± SE (n = 3–4 in A and B, n = 5–7 in C). Magnification: 40× scale bar: 50 μm. *P < 0.05 versus sham, #P < 0.05 versus Veh control.

in the hippocampal CA1 region after GCI, with an early peak of p-p38 at 30 min to 3 h post reperfusion and a later peak of p-p38 at 1 d post reperfusion. Other groups have also found post-ischemic activation of p38 in hippocampal CA1 neurons (Ferrer et al., 2003; Sun et al., 2010; Zhu et al., 2014), but most of these studies only focused on either early (e.g. 4 hours) or late (e.g. 2–3 days) reperfusion time points alone. It has been previously suggested that p38 activation in astrocytes and microglia after focal cerebral ischemia plays an indirect role in neuronal damage via elevation of neuroinflammatory cytokines, such as IL-1β and TNFα (Irving et al., 2000; Nito et al., 2012). While a similar mechanism is likely in play in GCI, our study suggests that neuronal activation of p38 may also play a direct role in GCI-induced delayed neuronal cell death via activation of the intrinsic apoptotic cell death pathway. In further support of this suggestion, inhibition of p38 activation has also been reported to reduce neuronal apoptosis in a model of chronic cerebral hypoperfusion (Yang et al., 2013). The current study also provides important information on the identity of the p38 isoforms that are regulated in the hippocampus and their role in pathology following GCI. Along these lines, our study yielded evidence that the p38α isoform is primarily responsible for the GCI-induced biphasic activation of p38 MAPK in the hippocampal CA1 region and that p38α activation contributes significantly to the ischemia-induced hippocampal neuronal cell death following GCI and reperfusion. P38β, in contrast, was not shown to undergo activation in the CA1 region after GCI. While the

factors regulating p38 activation in our study remain unclear, we propose that the transient p38α activation at the early reperfusion stage (30 min–3 h) may be due to rapid reactive oxygen species (ROS) production following ischemic reperfusion. In support of this possibility, we previously found that membrane NADPH oxidaseinduced superoxide anion (O 2 − ) production was significantly increased in the CA1 region of ovariectomized female rats 30 min–3 h after GCI (Zhang et al., 2009). In addition, NADPH oxidasederived ROS production is known to act upstream of p38 MAPK signaling in several cell types (Chan et al., 2005; Keshari et al., 2013; Kim et al., 2008; Lee et al., 2012; Rashed et al., 2011), and intriguingly, ROS have been shown to selectively activate the p38α isoform in these cells following a stressor (Bragado et al., 2007; Dolado et al., 2007; Kim et al., 2006). Thus, it is suggested that the selective activation of p38α observed 30 min–3 h after GCI in our study might be due to rapid, membrane-associated NADPH oxidase activation and ROS production. Our study also suggests that subcellular trafficking of activated p38 MAPK is critical for induction of apoptosis following cell stress. In addition to early nuclear trafficking of p38α after GCI, we also observed sustained phosphorylation/activation and mitochondrial translocation of p38α 24–48 h after GCI that was associated with well-known apoptotic events such as caspase cleavage, cytochrome c release, and loss of mitochondrial membrane potential 24–48 hours post GCI. Previous work has suggested that activation of the mitochondrial apoptotic pathway is a late reperfusion

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event after GCI (Carboni et al., 2005), a finding that is further supported by our observations. Interestingly, the activation of another stress-activated kinase, c-Jun-n-terminal kinase (JNK), has also been suggested to play a role in hippocampal CA1 neuronal death via a similar induction of mitochondrial-dependent apoptosis following GCI (Carboni et al., 2005; Davies and Tournier, 2012; Irving and Bamford, 2002). Like p38α, JNK also displays a late translocalization to the mitochondria after GCI (Chambers et al., 2013; Zablocka et al., 2003). Thus, activation and subcellular trafficking of p38 and JNK after reperfusion may play a critical role in apoptotic cell death after GCI. This suggestion is supported by our findings in the current study, as well as a previous study, which revealed that administration of either a p38α or a JNK inhibitor, respectively, provides significant neuroprotection against GCI (Zhang et al., 2008). With regard to upstream activators, a mechanistic connection between ROS induction and p38 activation and apoptosis was suggested previously in U937 cells in vitro where abolition of PLA2-induced ROS generation abrogated p38 MAPK activation and activation of the Fas/FasL cell death signal (Liu et al., 2009). Later work revealed that p38α MAPK directly induces up-regulation of Fas/FasL via nuclear translocation and subsequent activation of the downstream transcription factor ATF2 (Chen et al., 2009). Interestingly, the Fas signaling pathway is induced following cerebral ischemia and is known to be involved in the apoptotic neuronal death that occurs afterward (Liu et al., 2008; Shapira et al., 1990; Wang et al., 2006; Xiao et al., 2007). An additional major finding of the present study is that the steroid hormone E2 suppresses p38α MAPK activation in the hippocampus following GCI, which correlates with its neuroprotective effect. However, the mechanism whereby E2 regulates p38α activation is unclear. P38α protein expression in the CA1 region was unchanged by E2, suggesting that its effect is not at the transcriptional level. Moreover, in a previous study, we found that E2 also prevented the activation/phosphorylation of JNK in the CA1 region after GCI (Zhang et al., 2008). This is interesting, as both JNK and p38 MAPK are known to be dephosphorylated and inactivated by the dual phosphatase, mitogen activated kinase phosphatase-1 (MKP-1) (Caunt and Keyse, 2013). Preliminary studies by our group indicate that E2 strongly up-regulates expression of MKP-1 in the hippocampal CA1 region after GCI (Zhang et al., unpublished observation). Thus, E2 may suppress p38 MAPK (and JNK) activation after GCI by enhancing expression of the dual phosphatase, MKP-1. Further work is needed to explore this interesting possibility. It is currently unclear which E2 receptor mediates the E2 suppression of p38α MAPK. However, previous work by our group showed that the neuroprotective effect of E2 in the CA1 region after GCI was lost in ERα and GPR30 knockdown rats, but not in ERβ knockdown rats (Tang et al., 2014; Zhang et al., 2009). Thus, it is suggested that either ERα and/or GPR30 may mediate the p38α MAPK regulatory effect of E2. In support of this possibility, in vitro work by another group revealed that knockdown of ERα attenuated E2’s ability to suppress p38α MAPK activation in primary mouse neurons following treatment with a metabolic stressor (Barbati et al., 2012). Of significant interest, previous work by our lab provided evidence that extranuclear estrogen receptors are involved in the regulation and activation of multiple kinases and mediation of the anti-apoptotic and neuroprotective actions of E2 in the hippocampal CA1 region following GCI (Yang et al., 2010). This raises the possibility that extranuclear ERs may also contribute to the E2 inhibition of p38α activation and subcellular translocation that was observed in the present study. Further work is needed to address this possibility. In conclusion, the current study demonstrates that there is biphasic activation and subcellular translocation of p38α MAPK in the hippocampal CA1 region following GCI, which is critical for GCI-induced neuronal cell death. This study also provides evidence for the inhibitory effect of E2 on p38α MAPK activation and

subcellular translocation after GCI, which is suggested to contribute to the well-known anti-apoptotic, neuroprotective effect of E2 in the hippocampus. Acknowledgements This research was supported by a research grant (10SDG256009) from the American Heart Association to Q.Z. and the Foundation of President of Xuzhou Medical College (#2012KJZ16) to D. H. We would also like to extend our deepest gratitude to Darrell W. Brann for his academic mentorship and the use of his laboratory resources. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.mce.2014.11.010. References Barbati, C., Pierdominici, M., Gambardella, L., Malchiodi Albedi, F., Karas, R.H., Rosano, G., et al., 2012. Cell surface estrogen receptor alpha is upregulated during subchronic metabolic stress and inhibits neuronal cell degeneration. PLoS ONE 7, e42339. Barone, F.C., Irving, E.A., Ray, A.M., Lee, J.C., Kassis, S., Kumar, S., et al., 2001. Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia. Med. Res. Rev. 21, 129–145. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H.F., Marshall, C.J., 1998. Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol. 8, 1049–1057. Bi, X.Y., Wang, T.S., Zhang, M., Liu, Q.Q., Li, W.B., Zhang, Y., 2014. [The up-regulation of p-p38 MAPK during the induction of brain ischemic tolerance induced by intermittent hypobaric hypoxia preconditioning in rats]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 30, 97–100. Bragado, P., Armesilla, A., Silva, A., Porras, A., 2007. Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROS generation. Apoptosis 12, 1733–1742. Brann, D., Raz, L., Wang, R., Vadlamudi, R., Zhang, Q., 2012. Oestrogen signalling and neuroprotection in cerebral ischaemia. J. Neuroendocrinol. 24, 34–47. Brann, D.W., Dhandapani, K., Wakade, C., Mahesh, V.B., Khan, M.M., 2007. Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 72, 381–405. Carboni, S., Antonsson, B., Gaillard, P., Gotteland, J.P., Gillon, J.Y., Vitte, P.A., 2005. Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia. J. Neurochem. 92, 1054–1060. Cardaci, S., Filomeni, G., Rotilio, G., Ciriolo, M.R., 2010. p38(MAPK)/p53 signalling axis mediates neuronal apoptosis in response to tetrahydrobiopterin-induced oxidative stress and glucose uptake inhibition: implication for neurodegeneration. Biochem. J. 430, 439–451. Caunt, C.J., Keyse, S.M., 2013. Dual-specificity MAP kinase phosphatases (MKPs): shaping the outcome of MAP kinase signalling. FEBS J. 280, 489–504. Chambers, J.W., Pachori, A., Howard, S., Iqbal, S., LoGrasso, P.V., 2013. Inhibition of JNK mitochondrial localization and signaling is protective against ischemia/ reperfusion injury in rats. J. Biol. Chem. 288, 4000–4011. Chan, S.H., Hsu, K.S., Huang, C.C., Wang, L.L., Ou, C.C., Chan, J.Y., 2005. NADPH oxidase-derived superoxide anion mediates angiotensin II-induced pressor effect via activation of p38 mitogen-activated protein kinase in the rostral ventrolateral medulla. Circ. Res. 97, 772–780. Chen, K.C., Chiou, Y.L., Chang, L.S., 2009. JNK1/c-Jun and p38 alpha MAPK/ATF-2 pathways are responsible for upregulation of Fas/FasL in human chronic myeloid leukemia K562 cells upon exposure to Taiwan cobra phospholipase A2. J. Cell. Biochem. 108, 612–620. de Nadal, E., Ammerer, G., Posas, F., 2011. Controlling gene expression in response to stress. Nat. Rev. Genet. 12, 833–845. Davies, C., Tournier, C., 2012. Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies. Biochem. Soc. Trans. 40, 85–89. Dolado, I., Swat, A., Ajenjo, N., De Vita, G., Cuadrado, A., Nebreda, A.R., 2007. p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell 11, 191–205. Dreixler, J.C., Barone, F.C., Shaikh, A.R., Du, E., Roth, S., 2009. Mitogen-activated protein kinase p38alpha and retinal ischemic preconditioning. Exp. Eye Res. 89, 782–790. Ferrer, I., Friguls, B., Dalfo, E., Planas, A.M., 2003. Early modifications in the expression of mitogen-activated protein kinase (MAPK/ERK), stress-activated kinases SAPK/JNK and p38, and their phosphorylated substrates following focal cerebral ischemia. Acta Neuropathol. 105, 425–437. Ghatan, S., Larner, S., Kinoshita, Y., Hetman, M., Patel, L., Xia, Z., et al., 2000. p38 MAP kinase mediates bax translocation in nitric oxide-induced apoptosis in neurons. J. Cell Biol. 150, 335–347.

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