- Research article
- Open Access
PI3K/Akt-independent negative regulation of JNK signaling by MKP-7 after cerebral ischemia in rat hippocampus
- JianXi Zhu†1, 2,
- Wei Shen†3,
- Li Gao3,
- Hao Gu3,
- ShuTong Shen4,
- Yi Wang1, 2,
- HuiWen Wu4 and
- Jun Guo1, 2, 4Email author
© Zhu et al; licensee BioMed Central Ltd. 2013
- Received: 17 May 2012
- Accepted: 26 December 2012
- Published: 2 January 2013
The inactivation of c-Jun N-terminal kinase (JNK) is associated with anti-apoptotic and anti-inflammatory effects in cerebral ischemia, which can be induced by an imbalance between upstream phosphatases and kinases.
Mitogen-activated protein kinase phosphatase 7 (MKP-7) was upregulated significantly at 4 h of reperfusion postischemia in rat hippocampi. By administration of cycloheximide or siRNA against mitogen-activated protein kinase phosphatase 7 (MKP-7) in a rat model of ischemia/reperfusion, an obvious enhancement of JNK activity was observed in 4 h of reperfusion following ischemia, suggesting MKP-7 was involved in JNK inactivation after ischemia. The subcellular localization of MKP-7 altered after ischemia, and the inhibition of MKP-7 nuclear export by Leptomycin B up-regulated JNK activity. Although PI3K/Akt inhibition could block downregulation of JNK activity through SEK1 and MKK-7 activation, PI3K/Akt activity was not associated with the regulation of JNK by MKP-7.
MKP-7, independently of PI3K/Akt pathway, played a key role in downregulation of JNK activity after ischemia in the rat hippocampus, and the export of MKP-7 from the nucleus was involved in downregulation of cytoplasmic JNK activity in response to ischemic stimuli.
- Cerebral ischemia
Ischemia/reperfusion brain damage is a major risk factor of a variety of serious human neurological disorders such as learning disabilities, cerebral palsy, epilepsy and seizures or even death . c-Jun N-terminal kinase (JNK) is a potent mediator of inflammation and apoptosis . After ischemia, the JNK signaling pathway is highly activated, promoting the transcription of ischemia related genes and ultimately resulting in the lesion and dysfunction of neurons [3, 4]. Blockade of the JNK signaling pathway has been shown to protect neurons from ischemia/reperfusion injury and promote neuronal survival [5, 6]. However, rapid inactivation of JNK following its activation by cerebral ischemia has also been observed . Therefore, studying JNK inactivation may reveal the mechanism underlying the regulation of JNK activity after ischemia and support a new approach for treating ischemia/reperfusion injury.
The JNK signaling cascade is regulated by the balance between upstream kinases and phosphatases . The JNK pathway is a mitogen-activated protein kinase (MAPK) cascade in which mixed lineage kinases (MLKs/MAPKKK) activate MAP kinase kinase 4/7 (MKK4/7), which then activate JNK . Inhibiting MLK3 and MKK4/7 to block upstream kinase cascades involves JNK inactivation. Akt, known as a neuroprotective protein, can downregulate JNK activity through blocking an upsteam kinase . Akt inhibits the ASK1–SEK1–JNK2 signal transduction pathway by inactivating SEK1 through Ser80 phosphorylation in response to glucose deprivation . It has also been reported that the PI3K/Akt cascade negatively regulates the JNK pathway in PC12 cells by inhibiting MLK3 to downregulate MKK-7 . Meanwhile, the activity of Akt is upregulated by ischemic pre-treatment and estrogen, which is commonly considered to have a protective role in ischemia induced injury [13, 14].
Phosphatases also regulate JNK activity by direct dephosphorylation at residues Thr185 and Tyr183 . Members of the MKP family, which includes ten proteins, dephosphorylate MAPKs at both phosphothreonine and phosphotyrosine residues simultaneously within the MAPK TXY (Thr-Xaa-Tyr) activation motif and share similar structural folding at the catalytic site, as well as contain critical kinase-interacting motifs (KIMs) conferring specific MAPK substrate specificity . Individual MKPs generally show a substrate preference for one or more of the MAPKs. Among the MKPs, MKP-7 has a higher substrate specificity for JNK than for extracellular signal-regulated protein kinase (ERK) and P38 .
Some studies have demonstrated that the RNA interference (RNAi)-mediated ablation of MKP-7 increases the extent and duration of JNK activation induced by H2O2 stimulation in 293 T cells [18, 19], while overexpression of MKP-7 in COS-7 cells blocks the activation of JNK in a dose-dependent manner . Just as MAPKs are regulated by MKPs, in turn MKPs are also regulated by multiple factors. MKP-7 possesses a long C-terminal stretch containing both a nuclear export signal (NES) and a nuclear localization signal (NLS), which mediate their subcellular localization and nuclear-cytoplasmic shuttling by transport proteins . Acute oxidative stress has been reported to lead to redistribution of MKP-7 from the nucleus into the cytoplasm and a reduction in cytoplasmic p-JNK .
In this study, the relationships between upstream kinases and phosphatases were explored to determine the key regulator in JNK inactivation following cerebral ischemia. The findings of this study suggest that MKP-7 plays a key role in JNK inactivation during and after ischemia, and this regulation occurs independently of PI3K/Akt pathway.
Dual-phase phosphorylation of JNK induced by cerebral ischemia coincides with Akt-induced SEK1 and MKK-7 phosphorylation in the rat hippocampus
PI3K/Akt inhibitor LY294002 increases SEK1 and MKK-7 activities but does not result in upregulation of JNK activity following cerebral ischemia
The results above showed that Akt phosphorylation was associated with the JNK signaling cascade. However, to determine the exact role played by Akt in the JNK cascade in cerebral ischemia, the inhibitor LY294002 (LY) was employed to block the PI3K/Akt pathway. Rats underwent four-vessel occlusion and endured a 10-min period of ischemia, followed by reperfusion for 1 h. Phospho-specific antibodies were employed, and changes in JNK, Akt, SEK1 and MKK-7 activities were examined in the hippocampus of rats after the administration of LY or vehicle. The inactivated form of SEK1 is phosphorylated at Ser80, while the activated form of MKK7 is phosphorylated at Ser 271/Thr275. As shown in Figure 1B, in the 1-h reperfusion groups, a significant decline of p-Akt (Ser473) and p-SEK1 (Ser80) was observed in those rats treated with the LY compared with the vehicle-treated rats (P < 0.05). Meanwhile, the level of p-MKK-7 (Ser271/Thr275) was significantly elevated in the LY group compared with the vehicle group. However, the levels of activated JNK differed little between the 1-h reperfusion groups with and without the administration of LY. This result suggests that the PI3K/Akt inhibitor LY could downregulate Akt activity following cerebral ischemia, which increased the activity of SEK1 and MKK-7 but did not lead to the elevation JNK activity. Therefore, we hypothesized that another mechanism must be involved in JNK inactivation at 1 h reperfusion postischemia.
MAPK phosphatase is involved in JNK inactivation following cerebral ischemia
MKP-7 participates in JNK inactivation in the rat hippocampus after cerebral ischemia
Ischemia/reperfusion induces MKP-7 nuclear export to downregulate JNK activity
As it was demonstrated above that nuclear export of MKP-7 increased its cytoplasmic levels at 4 h of reperfusion after ischemia, we further examined whether this export of MKP-7 was involved in JNK inactivation. LMB, a specific inhibitor of nuclear export that blocks binding between the NES and export receptor, causes nuclear accumulation of MKP-7 . In this study, LMB was employed to identify the relationship between MKP-7 export and down-regulation of JNK activity. As shown in Figure 5B, there was a striking decrease in cytoplasmic MKP-7 and an increase in nuclear MKP-7 in the LMB group after 4 h of reperfusion. Meanwhile, as expected, the levels of JNK activity increased in the LMB group compared with the vehicle group. These results suggested that the export of MKP-7 was involved in JNK inactivation after 4 h of reperfusion after ischemia.
Ischemia-induced activation of MKP-7 is independent of the PI3K/Akt pathway
Nerve cells involved in ischemia/reperfusion injury activate a complex system of signal transductions, in which activation of JNK plays an important role in cerebral lesion. Inactivation of JNK blocks phosphorylation of a wide range of transcription factors and decreases the expression of ischemia related proteins . Therefore, the therapeutic potential of JNK inhibition is being investigated as an approach to protect cells from neurodegeneration and dysfunction .
Akt is an important cellular survival protein that protects cells from damage by inactivating JNK . During ischemia/reperfusion, ischemic pre-conditioning and estrogen may activate Akt to protect neurons by downregulating JNK activity. Therefore, Akt is considered an important negative regulator of JNK activity . However, in the current study, the evolution of Akt activity at 1 h of reperfusion did not result in JNK inactivation after ischemia and downregulation of Akt activity by PI3K/Akt inhibitor also did not induce increment of JNK activity at 1 h reperfusion. On the other hand, increases in the activity and level of MKP-7 at 4 h of reperfusion were observed and associated with JNK inactivation. Therefore, we propose that MKP-7 is an important newly defined regulator in the downregulation of JNK activity after ischemia.
The MKP-7 involves in the downregulation of JNK activity after ischemia in a PI3K/Akt independent manner. So it could be supposed that there are some differences between MKP-7 and Akt in their protective roles through JNK inactivation after ischemia. The increase of Akt activity to block the JNK cascade after ischemia is induced by external stimuli such as estrogen, anesthetic pre-conditioning and ischemic pre-conditioning [13, 29, 30]. However, the elevations in MKP-7 level and activity to inactivate JNK are induced by ischemia/reperfusion and not by other protective factors . In contrast to the protection mechanism of Akt, the inhibition of JNK by MKP-7 provides a negative feedback regulatory mechanism which prevents excessive ischemia/reperfusion injury after ischemia. Essentially, ischemia/reperfusion induces a series of alterations in physiological conditions and activates the JNK signal cascade. Meanwhile, it also elevates the level and activity of MKP-7, which cause a feedback signal to inhibit JNK activity by direct dephosphorylation. While blockade of the JNK cascade induced by Akt is long-lasting and can alleviate ischemia/reperfusion injury , the administration of siRNA to specifically target MKP-7 showed no effect on JNK activity after 24 h of reperfusion. These results suggested that the inactivation of JNK by MKP-7 did not last throughout the 24 h after reperfusion. Therefore, the protective effect of MKP-7 may have been short-lived and could not substantially alleviate ischemia/reperfusion injury following cerebral ischemia.
The regulation of phosphatases can usually be observed as a change in protein levels, phosphatase activity and stabilization. Elevation of MKP-7 mRNA by serum has been shown to result in the increase of the MKP-7 protein level . However, in this study, we found other regulatory mechanisms which elevated MKP levels. MKP-7 is a shuttle protein, and the exclusion of JNK-specific MKP-7 from the nucleus and its accumulation in the cytoplasm showed that it was exported from the nucleus after ischemia in the rat hippocampus. Since MKP-7 is a specific phosphatase of JNK, the change of its subcellular localization could have altered the level of p-JNK. Therefore, it is reasonable to conclude that the elevation of cytoplasmic MKP-7 after export from the nucleus ultimately led to the downregulation of JNK activity. This novel observation suggested that the altered subcellular localization of MKPs also led to the elevation of cytoplasmic MKP levels.
Animal surgery was performed in compliance with the Institutional Animal Care and Use Committee, conforming to international guidelines on the ethical use of animals, and was approved by the Biological Research Ethics Committee of Nanjing Medical University (permit number: NYLL2010-0009). Every effort was made to minimize the number of animals used and their suffering. Adult male Sprague–Dawley rats weighing 250–300 g (obtained from the Experimental Animal Center of Nanjing Medical University) were maintained at room temperature and given free access to food and water. Before surgery, the animals were deprived of food overnight. The rats were anesthetized by intraperitoneal injection of 20% chloral hydrate (250 mg/kg) and surgically prepared for four-vessel occlusion according to the method described by Pulsinelli et al. . Bilateral vertebral arteries were electrocauterized, and both common carotid arteries were dissected free. Twenty-four hours later, both common carotids were occluded with aneurysm clips. Rats were kept awake during the periods of ischemia and subsequent reperfusion, which eliminated the potential effects of sedatives such as chloral hydrate on the results of the experiment. The selected rats matched the following criteria: maintenance of dilated pupils, loss of cornea reflex, completely flat electroencephalogram, rigor of the extremities and vertebral column, and maintenance of rectal temperature at approximately 37°C. Rats in the sham group received the same surgical procedures except for the occlusion of the four arteries.
Infusion and administration of drugs
The phosphoinositide-3 kinase (PI3K)/Akt inhibitor LY294002 (LY, 10 mM, Calbiochem-Novabiochem Corp., San Diego, CA, USA), MAPK phosphatase or protein synthesis inhibitor cycloheximide (CHX, 177 mM, Sigma-Aldrich, St. Louis, MO, USA), the Dusp6 inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro- 1H-inden-1-one (BCI, 28 mM, Sigma-Aldrich Co.) and MKP-7 nuclear export inhibitor Leptomycin B (LMB, 0.2 μg/μl, Enzo Life Sciences, Farmington, NY, USA) or the same volume of vehicle (2 μL) were injected into the cerebral ventricle (0.8 mm posterior and 1.5 mm lateral to the bregma; 3.5 mm deep) using a micro-injector 30 min before ischemia induction according to previous previously published reports [34, 35]. The injector was retained in place for another 5 min after the injection, avoiding any possible backflow of liquid along with the injection void.
In vivoMKP-7 siRNA transfer
Five microliters of MKP-7 siRNA (20 μM, RiboBio Co., Guangzhou, China) or control siRNA-A (20 μM, RiboBio) was diluted with the same volume of transfection reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After gently pipetting the solution up and down, the mixture was then incubated for 45 min at room temperature (~25°C) and injected into the cerebral ventricle (0.8 mm posterior and 1.5 mm lateral to the bregma; 3.5 mm deep) using a micro-injector. This injection was repeated four times, every 12 h starting 2 days before ischemia induction .
Tissue sample preparation
Rats were sacrificed by decapitation at various times: 10 min after ischemia and 15 min, 1, 2, 4, 6 and 24 h after reperfusion. The hippocampal tissues were removed on ice and homogenized in 1:10 (W/V) ice-cold homogenization buffer A [50 mM HEPES (pH 7.4), 100 mM KCL, 1 mM Na3VO4, 50 mM NaF and 1 mM PMSF] containing 1% mammalian proteinase inhibitor cocktail (Sigma-Aldrich). Proteins in the cytoplasm and membrane were extracted after centrifugation at 800 × g and 4°C. After this step, the supernatant was centrifuged at 14,000 × g and 4°C to harvest cytoplasmic proteins. The resulting pellet after the first centrifugation was resuspended in homogenization buffer B [50 mM HEPES (pH7.4), 100 mM KCL, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 mM DTT, 1% cocktail, and 5% SDS] and kept on ice for 30 min, followed by ultrasonic disruption for 6 s, repeated 6 times and then centrifugation at 14,000 × g and 4°C. The supernatants containing the nuclear or cytoplasmic proteins were extracted and then stored at −80°C until assayed. The protein concentrations of the extracts were determined according to the Bradford method using bovine serum albumin (BSA) as a standard .
Denatured samples were separated by 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes (NC, pore size, 0.2 μm). The proteins on the membrane were probed with primary antibodies against JNK, phosphorylated JNK at Thr183/Tyr185, Akt, phosphorylated Akt at Ser473, phosphorylated SEK1 at Ser80, phosphorylated MKK7 at Ser271/Thr275, MKP-7 (Santa Cruz Biotechnology) and β-actin (Boster Biotechnology, WuHan, HB, China) at 4°C overnight. Following three 5–10 min washes in Tris-buffered saline, 0.1% Tween-20 (TBST), the immune complexes on the membrane were then incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or mouse secondary antibody (1:5000, ZhongShan Golden Bridge Biotechnology, Peking, China) for 2 h. Following the incubation, the membranes were then washed in TBST three times for 10 min each time. The bands on the membranes were scanned and analyzed using an image analyzer.
Phosphatase activity assay
The primary rabbit antibody to MKP-7 (1–2 μg) was added to each sample of total proteins (200 μl). After shaking overnight at 4°C, protein A/G PLUS-Agarose (10 μl, Santa Cruz Biotechnology) was added and incubated with shaking for 2 h at 4°C. The protein A/G PLUS-Agarose was precipitated by centrifugation at 5000 × g and washed three times with immunoprecipitation buffer. The washed agarose was resuspended in 200 μl phosphatase assay buffer containing 20 mM pNPP and 50 mM imidazole (pH 7.5) (both from Enzo Life Sciences, Farmington, NY, USA) and incubated for 2 h at 30°C. The reaction was stopped by the addition of 800 μl of 0.25 N NaOH, and p-NPP hydrolysis was measured by absorbance at 410 nm.
Data and statistical analysis
Data were documented as means ± SD from at least three independent rats. One-way analysis of variance (ANOVA) followed by Duncan’s new multiple range method was applied to analyze the statistical results. P-values < 0.05 were considered significant.
The work was supported by grants from the National Natural Science Foundation of China (No. 81170714), the Program for Science and Technology of Nanjing (No. 200901081) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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