Dual-phase phosphorylation of JNK induced by cerebral ischemia coincides with Akt-induced SEK1 and MKK-7 phosphorylation in the rat hippocampus

It is well known that cerebral ischemia highly induces activation of the JNK pathway through phosphorylation, leading to apoptosis and dysfunction of neurons. However, JNK activity has been observed to obviously decrease following its activation by cerebral ischemia [7], but the precise mechanism remains unclear. To determine the underlying cause of the abrupt JNK inactivation following cerebral ischemia, phosphorylation of JNK at Thr183/Tyr185 and its upstream molecules Akt at Ser473, SEK1 at Ser80 and MKK-7 at Ser 271/Thr275 were examined in the rat hippocampus following ischemia. Rats were subjected to four-vessel occlusion, with 10 min of ischemia followed by reperfusion for 15 min, 1, 2, 4, 6 and 24 h. As shown in Figure 1A, phosphorylation of JNK showed a biphasic pattern of elevation during reperfusion, gradually increasing from 15 min (P < 0.05), peaking at 1 h (P < 0.05), but then decreasing significantly at 4 h (P < 0.05) before elevating again at 24 h (P < 0.05). Meanwhile, the phosphorylation of Akt, SEK1 and MKK-7 peaked at 1 h (P < 0.05), showing that phosphorylation of these proteins may be associated with JNK. After peaking at 1 h, their phosphorylation sharply decreased at 4 h (P < 0.05) and was elevated again after 24 h (P < 0.05). Meanwhile, no changes were observed in the levels of total JNK, Akt, SEK1, MKK-7 and β-actin in each group during the reperfusion (P > 0.05, Figure 1). These results demonstrated that cerebral ischemia induced the dual-phase phosphorylation of JNK, and the phosphorylation patterns of Akt, SEK1 and MKK-7 showed the same trends as that of JNK after ischemia.

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

Activated JNK promotes ischemic injury related protein expression through phosphorylation of transcription factors such as c-jun. In order to control appropriate gene transcription, the activity of JNK must be tightly regulated by the actions coordinated between protein kinases and phosphatases. MKPs comprise a subset of protein tyrosine phosphatases, which can dephosphorylate both phosphothreonine and phosphotyrosine residues. Cycloheximide (CHX) is usually considered a protein synthesis inhibitor, and some studies have also shown that it can inhibit the activity of MKPs [22]. To explore whether MKPs were involved in JNK inactivation after ischemia in the rats, CHX was employed in this study. As shown in Figure 2A, levels of JNK activity were significantly elevated in the CHX group compared with the vehicle group at 4 h after reperfusion (P < 0.05). BCI, an allosteric inhibitor of Dusp6 [23], was also used in this study, however, the levels of JNK activity were not changed in the BCI group compared with the vehicle group at 4 h after reperfusion (Figure 2B, P > 0.05). Furthermore, activated JNK levels differed little between the 24-h reperfusion groups with and without the administration of MKPs inhibitor (CHX or BCI), with both groups exhibiting high levels (P > 0.05). This result suggested that a few MKPs but not Dusp6 were involved in JNK inactivation at 4 h reperfusion after ischemia.

MKP-7 participates in JNK inactivation in the rat hippocampus after cerebral ischemia

We wanted to further identify which member of the MKP family was involved in the downregulation of JNK activity at 4 h after reperfusion. MKP-7 is a JNK-specific phosphatase. A previous study showed that the level of JNK phosphorylation is significantly prolonged when the expression of endogenous MKP-7 is ablated by siRNA [18, 19]. In order to explore the role of MKP-7 in JNK inactivation after ischemia, its cytoplasmic level and activity were observed after ischemia/reperfusion. As shown in Figure 3A, the levels of cytoplasmic MKP-7 reached peak levels at 4 h (P < 0.05). However, it decreased obviously at 24 h again (P < 0.05). This result showed that the trend of cytoplasmic MKP-7 protein level followed the opposite pattern as that of JNK activity after ischemia. As shown in Figure 3B, MKP-7 activity after ischemia was also observed to slightly increase from 15 min to 2 h (P < 0.05) and reached a peak level at 4 h (P < 0.05). However, it decreased significantly at 24 h (P < 0.05). The trend in MKP-7 activity was similar to that of the corresponding cytoplasmic protein level and again displayed the opposite pattern to that of JNK activity.

The results presented above indicated that MKP-7 could participate in the inhibition of JNK activity after ischemia. To further clarify the role of MKP-7 in down-regulation of JNK activity after ischemia, MKP-7 siRNA was employed to decrease the amount of cytoplasmic MKP-7. As shown in Figure 4, JNK activity was markedly reduced in the 4-h reperfusion control and 4-h reperfusion vehicle groups compared with that in the 1-h reperfusion control. As expected, this inhibitory effect was sufficiently relieved after 4 h of reperfusion regardless of MKP-7 siRNA treatment. However, in contrast with the changes in p-JNK, this phosphorylation state was maintained after 24 h of reperfusion, and administration of MKP-7 siRNA was unable to reverse it (P > 0.05). This result confirmed our hypothesis that MKP-7 was involved in JNK inactivation during the 4 h but not 24 h of reperfusion post-ischemia.

Ischemia/reperfusion induces MKP-7 nuclear export to downregulate JNK activity

The results above showed that ischemia/reperfusion elevated the levels and activities of cytoplasmic MKP-7 to downregulate JNK activity at 4 h of reperfusion after ischemia. The next step was to investigate the potential mechanism of MKP-7 upregulation after ischemia. MKP-7 contains an intrinsic NES motif which mediates subcellular localization, and allows this protein to function as a nuclear-cytoplasmic shuttle protein upon stimulation by extracellular signals. To explore whether MKP-7 subcellular localization was altered during ischemia/reperfusion, the cytoplasmic and nuclear levels of MKP-7 were examined by western blot. As shown in Figure 5A, there was a striking increase in cytoplasmic MKP-7 and a decrease in nuclear MKP-7 in the 4-h reperfusion group compared with the sham group. However, cytoplasmic MKP-7 obviously decreased, while nuclear MKP-7 was strikingly elevated after 24 h of reperfusion. The results above suggested that MKP-7 was transported from the nucleus to cytoplasm at 4 h of reperfusion and was imported back to the nucleus at 24 h of reperfusion after ischemia.

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 [21]. 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

We also found that treatment with the PI3K/Akt inhibitor LY294002 induced a significant elevation in JNK activity during the 4 h of reperfusion (Figure 6A). Although there is much evidence to support the contention that JNK dephosphorylation/inactivation by MKP-7 is independent of PI3K/Akt pathway, some studies have suggested that PI3K/Akt cascade plays a key role in the downregulation of JNK activity [12, 24]. Furthermore, whether PI3K/Akt pathway downregulates JNK via MKP-7 was previously unknown. In order to identify the relationship between inactivation of JNK by MKPs and the PI3K/Akt signaling pathway, the level and activity of MKP-7 were examined by administration of the PI3K/Akt inhibitor LY294002 (LY). As shown in Figure 6B, there was no difference between the LY and vehicle groups in terms of MKP-7 activity during the 4-h reperfusion. Meanwhile, the level of MKP-7 also differed little between the 4-h reperfusion groups with and without the administration of LY, both exhibiting high levels (Figure 6C). Furthermore, the protein level and activity of JNK and MKP-7 kept stable between the sham groups with and without the administration of LY. These results suggested that the MKP-7 downregulation of JNK activity after ischemia did not depend on the PI3K/Akt pathway.

Surgical procedure

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. [33]. 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 [36].

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 [37].

Western blot

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.