brain research 1566 (2014) 60–68
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Research Report
Assembly of the FKBP51–PHLPP2–AKT signaling complex in cerebral ischemia/reperfusion injury in rats Xiu-E Weia,n, Feng-Yu Zhangb, Kai Wanga, Qing-Xiu Zhanga, Liang-Qun Ronga a
The Second Affiliated Hospital of Xuzhou Medical University, Xuzhou 221000, China Liaocheng Hospital, Liaocheng 252000, China
b
ar t ic l e in f o
abs tra ct
Article history:
The imbalance of cell pro-death and pro-survival signaling pathways determines the neuronal
Accepted 8 April 2014
fate during cerebral ischemia/reperfusion (I/R) injury. However, the biological mechanisms
Available online 16 April 2014
regulating the balance between activation of the pro-death or the pro-survival signaling
Keywords:
pathways remain unclear. In this study, a rat model of I/R injury was established using four-
FKBP51–PHLPP–AKT signaling
vessel occlusion followed by different times of reperfusion. I/R injury did not affect the level of
complex
FK506 binding protein 51 (FKBP51), PH domain and leucine rich repeat protein phosphatases
PHLPP2
(PHLPP)-2, and AKT, but induced assembly of the FKBP51-PHLPP2-AKT signaling complex, as
p-AKT
indicated by the enhancement of interactions among these compounds following reperfusion.
p-JNKs
Using an antisense oligonucleotide, PHLPP2 expression was effectively inhibited. Critically, the
Cell death
inhibition of PHLPP2 prohibited the interactions of FKBP51, PHLPP2 and AKT, reversed the decrease of p-AKT expression and increased the expression of p-JNKs and p-c-Jun elicited by I/R injury. In addition, PHLPP2 inhibition reversed I/R-injury-induced Caspase-3 cleavage and loss of pyramid neurons in the CA1 region of hippocampus. The results of the current study indicate that the assembly of the FKBP51-PHLPP2-AKT signaling complex plays a critical role in mediating cell death in I/R injury. The inhibition of PHLPP2 via antisense oligonucleotide treatment may be an effective method to prohibit the assembly of the FKBP51–PHLPP–AKT signaling complex, thus balancing the cell pro-survival and pro-death signaling pathways ultimately mitigating cell death in I/R injury. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke is one of the most severe cerebrovascular diseases endangering human health. In conjunction with high n
incidence rates, serious sequel, high morbidity and recurrence rates affect the disease therapy accordingly (Goldstein et al., 2006). Therefore, establishing a potential therapeutic target for ischemia/reperfusion (I/R) injury is of high importance. Recently,
Correspondence to:Meijian Road 32, Quanshan District, Xuzhou, Jiangsu, China. Fax: þ86 516 85326115. E-mail address: [email protected] (X.-E. Wei).
http://dx.doi.org/10.1016/j.brainres.2014.04.009 0006-8993/& 2014 Elsevier B.V. All rights reserved.
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the pathophysiological pathways of stroke have become of high importance in the study of cerebrovascular disease (Deb et al., 2010). A line of hypothesis on the occurrence, development and prognosis of stroke have previously been proposed (Woodruff et al., 2011), however the balance between the cell survival and cell death signaling pathways, both of which play critical roles in determining neuron survival (Abe et al., 2000; Vassalli et al., 2012), is yet to be explored. Under pathological conditions, it has been reported that the balance between these two signaling pathways is disrupted, ultimately resulting in neuronal injury or cell death (Morrison et al., 2002). The PH domain and leucine rich repeat protein phosphatases (PHLPP) can specifically dephosphorylate the hydrophobic motif, Ser-473 in AKT, thus partially inactivating the kinase (Gao et al., 2005). There are three major subtypes included in the PHLPP2 family, namely PHLPP1α, PHLPP1β and PHLPP2 all with structural similarity including: a putative Ras association domain; a pleckstrin homology domain; a series of leucine-rich repeats; a PP2C phosphatase domain and a C-terminal PDZ ligand. As PHLPP negatively regulates the pro-cell survival protein kinase B (PKB)/ AKT pathway, this phosphatase has recently attracted attention in several lines of research areas including in tumor progression (Gao et al., 2005), diabetes (Andreozzi et al., 2011) and I/R injury (Jackson et al., 2013; Miyamoto et al., 2010). It has been reported that PHLPP is widely expressed in the cytoplasm, the nuclei and the cell membrane of breast, prostate, ovarian and brain tissue (Gao et al., 2005). Importantly, low expression of PHLPP1 has been reported to have potential protective effects against cardiomyocyte or neuron damage (Miyamoto et al., 2010; Shiojima and Walsh, 2006). The exact biological roles of PHLPP2 in cerebral I/R injury currently remain unknown. Both AKT1 and AKT3 are widely expressed in the brain and play critical roles in lowering
cerebral injury (Xu et al., 2012). In addition, PHLPP2 has been reported to negatively regulate AKT1 and AKT3 dephosphorylation (Brognard et al., 2007). The manner in which PHLPP2 regulates p-AKT and the downstream pathways during cerebral I/R injury remains unknown. The FK506 binding protein 51 (FKBP51) not only enhances the interaction between PHLPP and AKT but also increases PHLPP activity, leading to AKT dephosphorylation (Pei et al., 2009; Wang, 2011). The current study aimed to investigate the hypotheses that I/R injury facilitates the assembly of the FKBP51–PHLPP2–AKT signaling complex thus regulating the pro-cell survival and cell death signaling pathways. Using a rat model of cerebral ischemia injury, the current study successfully demonstrated that the assembly of the FKBP51– PHLPP2–AKT signaling complex plays an essential role in mediating cell death during I/R injury. The inhibition of PHLPP2 through antisense oligonucleotide treatment was an effective method which can inhibit the assembly of the FKBP51-PHLPP2-AKT signaling complex and reverse I/R injury-induced disruption of the balance between the cell death and pro-cell survival signaling pathways. The results of the current study provide new initiatives in terms of new therapeutic treatments for I/R injury.
2.
Results
2.1. I/R injury induced assembly of FKBP51–PHLPP2–AKT signaling complex Consistent with previous studies, western blot analyses confirmed that PHLPP2 and FKBP51 proteins were expressed in rat
IB:PHLPP2
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Fig. 1 – Ischemia/reperfusion (I/R) injury did not affect PHLPP2 and FKBP51 expression. (A) Representative blots of PHLPP2 expression after different times of reperfusion. (B) Quantification data of PHLPP2 expression. (C) Representative blots of FKBP51 expression after different times of reperfusion. (D) Quantification data of FKBP51 expression. The data were shown in Mean and SEM with six repeats.
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hippocampal tissue (Brognard et al., 2007; Jackson et al., 2010). In addition, compared with sham control, the protein expression of both PHLPP2 and FKBP51 at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h and 3 days after reperfusion were not significantly different (Fig. 1). The interactions between FKBP51, PHLPP2 and AKT proteins were determined using immunoprecipitation (IP). As shown in Fig. 2, FKBP51, PHLPP2 and AKT proteins were immunoprecipitated together, indicating interactions between these three proteins in rat hippocampal tissue. Critically, the interactions of these three proteins gradually enhanced followed by a decrease with the increase of reperfusion time. A significant difference was observed after 6 h reperfusion compared with the sham control.
2.2. PHLPP2 inhibition reduced the assembly of the FKBP51–PHLPP–AKT signaling complex during I/R injury Three PHLPP2 antisense oligonucleotides (AS ODNs) and a missense oligonucleotides (MS ODNs) were designed to screen the antisense oligonucleotide which can block the PHLPP2 expression. As shown in Fig. 3A and B, AS ODNs (5' TTTCCAAACGGTTTCCTGC 3') significantly inhibited
IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 IP:PHLPP2 IB:Akt IB:β-actin
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2.3. PHLPP2 inhibition increased AKT phosphorylation following I/R injury It has previously been reported that the PKB/AKT pathway is important in the pro-cell survival pathway in neurons (Zhu et al., 2012). In contrast, in the current study, as shown in Fig. 4, I/R (6 h) significantly decreased the levels of p-AKT and did not affect the levels of total AKT. Both Tris–EDTA buffer (TE) and MS ODNs treatment did not influence the levels of p-AKT compared with I/R group. In contrast, AS ODNs treatment significantly increased p-AKT levels compared with MS ODNs treatment. These data indicate that inhibition of PHLPP2 can activate the PKB/AKT pathway most likely through preventing the assembly of the FKBP51–PHLPP2–AKT signaling complex.
2.4. Inhibition of PHLPP2 decreased phosphorylation of JNKs following I/R injury
IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 IP:PHLPP2 IB:Akt
O.D.(Folds vs sham)
PHLPP2 expression to approximately 30% of the control level. The PHLPP2 level in the hippocampus after AS ODNs treatment was significantly reduced compared with that after MS ODNs treatment. The influences of PHLPP2 inhibition on the assembly of the FKBP51–PHLPP2–AKT signaling complex was then investigated after I/R injury (6 h). The MS ODNs was used as the negative control. Compared with the MS ODNs, inhibition of PHLPP2 following AS ODNs treatment significantly reduced the assembly of the FKBP51–PHLPP2–AKT signaling complex as evidenced by the decreased interactions between these three proteins (Fig. 3C and D).
Fig. 2 – I/R injury promoted assembly of FKBP51–PHLPP2– AKT signaling complex. (A) The representative immunoprecipitation blots of FKBP51, PHLPP and AKT after different times of reperfusion. (B) Quantification data showing the interactions among these three proteins were increased with the increase of reperfusion time. The data were shown in Mean and SEM with six repeats. nPo0.05 compared with sham control.
It has been reported that activation of the PKB/AKT pathway can protect against ischemia injury by inhibiting the phosphorylation of JNKs either directly or indirectly (Zhang et al., 2007; Zhu et al., 2013). To address the effects of PHLPP2 inhibition on the JNK pathway, the activation of JNKs and c-Jun were investigated. In the current study, I/R injury did not affect total JNK (Fig. 5A and B) and c-Jun (Fig. 5C and D). The peak increase of p-JNKs, p-c-Jun was observed 6 h after reperfusion. Compared with the sham control, I/R (6 h) significantly increased the levels of p-JNKs. Both TE and MS ODNs treatment did not affect p-JNKs levels compared with the control group. Interestingly, AS ODNs treatment significantly decreased p-JNKs levels compared with MS ODNs treatment. The levels of p-c-Jun 6 h after reperfusion were then investigated. As shown in Fig. 5C and D, compared with sham control, I/R (6 h) significantly increased the level of p-c-Jun. Both TE and MS ODNs treatment did not affect the level of p-c-Jun compared with the control group. The AS ODNs treatment significantly decreased p-c-Jun level compared with the MS ODNs treatment group. In addition, the levels of apoptosis “effector” caspase-3 were determined in the different experimental groups. Compared with sham control, I/R (6 h) significantly increased the level of cleaved-caspase-3 level (Fig. 6). Neither TE nor MS ODNs treatment reversed the level of cleaved-caspase-3 compared with the control group. Treatment with the AS ODNs did however significantly decrease the level of cleavedcaspase-3 compared with MS ODNs treatment.
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IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 IP:PHLPP2 IB:Akt IB:β-actin
IB:PHLPP2 IB:β-actin
IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 2.0
O.D.(Folds vs sham)
1.0
0.5
IP:PHLPP2 IB:Akt
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P2 LP PH
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Fig. 3 – PHLPP2 inhibition using an antisense oligonucleotide prevented against assembly of FKBP51–PHLPP–AKT signaling complex observed 6 h after reperfusion. (A) Representative blots of PHLPP2 expression after antisense oligonucleotide treatment. (B) Quantification data of PHLPP2 expression after antisense oligonucleotide treatment. (C) The representative immunoprecipitation blots of FKBP51, PHLPP and AKT after PHLPP2 inhibition and I/R injury. (D) Quantification data showed the increased interactions among these three proteins after I/R injury were prevented by PHLPP2 inhibition. The data were shown in Mean and SEM with six repeats. *Po0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
2.5. Inhibition of PHLPP2 prevented neuronal death after I/R injury To demonstrate the effects of the assembly of the FKBP51– PHLPP2–AKT signaling complex on neuronal death after I/R injury, Cresyl violet staining was applied to cells to determine the number of pyramid neurons in the CA1 region. As shown in Fig. 7, the pyramid neurons in the sham control group were closely spaced, but not in I/R group, the MS ODNs and the TE groups. This was ameliorated by AS ODNs treatment. The losses of pyramid neurons were observed in I/R and MS ODNs group. However, the loss of pyramid neurons after I/R was significantly reversed with AS ODNs treatment.
3.
Discussion
The risk factors for ischemic stroke have been largely reported, including old age, high blood pressure, heart disease, smoking
and diabetes (Allen and Bayraktutan, 2008). The mechanisms involved in the initiation of ischemic stroke remain controversial with the widely accepted view being that is the disruption of the balance between neuronal cell survival and cell death responsible for ischemic stroke. Among these pathways, the PI3K/AKT and JNKs pathway are two primary pathways playing opposite roles in the determination of cell fate (Hui et al., 2005). Unfortunately, the upstream pathway mediating PI3K/AKT is not well known. Whilst the inhibition of PHLPP1 has been reported to regulate AKT phosphorylation and can significantly increase cell survival and diminish the infarct size after I/R injury (Chen et al., 2013), the exact roles of PHLPP2 in I/R injury is currently not well documented. In the present study, I/R injury did not affect the level of PHLPP2. Interestingly, in the current study, I/R injury promoted the assembly of the FKBP51– PHLPP2–AKT signaling complex. The immunoprecipitation between FKBP51, PHLPP2 and AKT were increased with the increase of reperfusion time. Supporting these findings is a report implicating that FKBP51 acts as a scaffolding protein for
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IB:Akt IB:p-Akt
IB:β-actin
IB:Akt IB:p-Akt
O.D.(Folds vs sham)
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Fig. 4 – PHLPP2 inhibition prevented against I/R injuryinduced decrease of p-AKT level. (A) Representative blots of p-AKT expression after antisense oligonucleotide treatment. (B) Quantification data of p-AKT expression after antisense oligonucleotide treatment. The data are shown as Mean7SEM with six repeats. nPo0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
AKT and PHLPP and promotes dephosphorylation of AKT in cancer etiology in response to chemotherapy (Pei et al., 2009). The results of the current study indicate that FKBP51 acted as a scaffold protein regulating AKT and PHLPP2 after cerebral I/R injury. The assembly of the FKBP51–PHLPP2–AKT signaling complex may be responsible for the activation of the cell death pathway and consequently cell death. Identifying the drugs that can block the assembly of this signaling complex may be therapeutically important for the prevention of stroke. As FKBP inhibitors are unlikely to inhibit the AKT– FKBP–PHLPP network (Fabian et al., 2013), PHLPP2 may be the possible therapeutic target protein. Inhibition of specific protein expression using AS ODNs is a useful strategy in different disease treatment (Chan et al., 2006). In the current study, the AS ODNs (5' TTTCCAAACGGTTTCCTGC 3') significantly inhibited the protein expression of PHLPP2 to approximately 30% of control levels and the efficiency rate of inhibition was high (70%). Importantly, this AS ODNs inhibited the assembly of the FKBP51–PHLPP2–AKT signaling complex, which appeared 6 h after reperfusion. After inhibition of PHLPP2, the interaction of FKBP51 with AKT during I/R injury was decreased. It is may be postulated that interaction
between FKBP51 and PHLPP2 was reduced due to the decreased expression of PHLPP2. Surprisingly, with the similar amount of input protein PHLPP2, the immunoprecipitated AKT levels were reduced after AS ODNs treatment following I/R injury. These data suggest that the interaction between PHLPP2 and AKT was decreased most likely through the decreased scaffolding effect of FKBP51. Whilst the direct effect of PHLPP2 on AKT dephosphorylation was not excluded from the current study, unaltered PHLPP2 expression during I/R injury partially explained the important role of the assembly of the FKBP51–PHLPP2–AKT signaling complex, rather than the actual level of PHLPP2 itself following I/R injury. Comparably, β-arrestin 1 has been reported to specifically scaffold PHLPP2 and Akt1 to regulate phosphorylation of Akt (Crotty et al., 2013). That study and ours emphasized the importance of signaling complex in regulating cell prosurvival or pro-death pathways. The inhibition of PHLPP2 using AS ODNs may be an effective way to prevent I/R injury as such inhibition in the current study blocked the assembly of the FKBP51–PHLPP2– AKT signaling complex. To this end, the current study confirmed that PHLPP2 inhibition induces blockage of the assembly of the FKBP51–PHLPP–AKT signaling complex thus contributing to the promotion of neuronal cell survival or blockage of neuron death. The PI3K/AKT pathway is an important pro-cell survival pathway in mammalian cells under different pathological conditions (Datta et al., 1999; Zhu et al., 2012). The blockage of this pathway can lead to cell death, whereas activation of this pathway prevents cell death. Consistently, in this study, the level of p-AKT was significantly decreased, indicating that the PI3K/AKT pathway was inhibited after I/R injury. Knockdown of FKBP1 or the TRP domain has been reported to attenuate the interaction between AKT and PHLPP (Pei et al., 2009). The inhibition using the PHLPP2 AS ODNs further supports this notion. In the current study, PHLPP2 inhibition compromised the interactions of these three proteins and in turn, the level of p-AKT was enhanced in the experimental group compared with I/R group. These data indicate that p-AKT level is possibly regulated by the FKBP51–PHLPP2–AKT signaling complex. The present study therefore clarifies a novel mediating mechanism of PHLPP2 on p-AKT after I/R injury. Unlike the PI3K/AKT pathway, the JNKs pathway plays an opposing role in the mediation of cell death (Shen and Liu, 2006). The activation of the JNKs pathway can indeed exacerbate neurodegeneration (Borsello and Forloni, 2007). A scaffold protein, c-Jun NH2 terminal kinase interacting protein-1 (JIP-1) can activate JNK through direct interaction with JNK (Whitmarsh et al., 2001). However, interaction of AKT with JIP-1 compromises the interaction of JIP-1 with JNK, in turn inhibiting the activation of the JNKs pathway and antagonizing neuron apoptosis (Whitmarsh et al., 2001). Prior research has shown that AKT combined with POSH/MLK/MKK/JNK forms a mediating module, which can inhibit the activation of the JNK pathway (Figueroa et al., 2003). In the current study, I/R injury activated the JNKs pathway, as indicated by increased levels of p-JNKs and increased phosphorylation of the nuclear substrate c-Jun. Concurrently, inhibition of PHLPP2 using the AS ODNs could inactivate the JNKs pathway. To further confirm the injury of I/R and the protection provided by PHLPP2 inhibition, the cleaved
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4.2.
Animals and model
Male Sprague-Dawley rats (weighing 220–250 g) were purchased from the Experimental Animals Center of Xuzhou Medical College (China). Transient forebrain ischemia was induced by four-vessel occlusion (4-VO) as previously described (Clemens et al., 1998). Briefly, rats were prepared for forebrain ischemia under 20% chloral hydrate (300–350 mg/kg) anesthesia by elec-
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The animal care and surgical procedures were performed under the established standards of the Second Affiliated Hospital of Xuzhou Medical College (China).
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Ethics statement
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3
I/R
Experimental procedures
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caspase-3 expression and cell loss in the CA1 region were determined. As expected, I/R injury-induced apoptosis pathway was inhibited by PHLPP2 inhibition as indicated by decreased cleaved caspase-3 level. In conclusion, the current study demonstrates that I/R injury does not affect FKBP51, PHLPP2 and AKT protein expression, but increases the assembly of the FKBP51–PHLPP2–AKT signaling complex (Fig. 8). PHLPP2 inhibition using a specific antisense oligonucleotide could effectively compromise assembly of the FKBP51–PHLPP2–AKT signaling complex, which most likely activates p-AKT and blocks the JNKs pathway, leading to neuronal protection. The current study provides a novel stratagem for stroke prevention and therapy.
Fig. 6 – PHLPP2 inhibition prevented against I/R injury-induced caspase-3 cleavage. (A) Representative blots of cleaved-caspase3 expression after antisense oligonucleotide treatment. (B) Quantification data of cleaved-caspase-3 expression after antisense oligonucleotide treatment. The data were shown in Mean and SEM with six repeats. *Po0.05 compared with sham control. #Po0.05 compared with missense oligonucleotides treatment.
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Fig. 5 – PHLPP2 inhibition prevented against I/R injury-induced activation of JNKs pathway. (A) Representative blots of p-JNK expression after antisense oligonucleotide treatment. (B) Quantification data of p-JNK expression after antisense oligonucleotide treatment. (C) Representative blots of p-c-Jun expression after antisense oligonucleotide treatment. (D) Quantification data of p-c-Jun expression after antisense oligonucleotide treatment. The data were shown in Mean and SEM with six repeats. *Po0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
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Fig. 7 – PHLPP2 inhibition prevented I/R injury-induced cell loss in CA1 region. (A) Representative image of hippocampus after Cresyl violet staining (scale bar: 100 lm). The black rectangle indicates the CA1 region. (B) The representative images of CA1 region in sham control, I/R 5d, TEþI/R 5 d, PHLPP2 MS ODNsþI/R 5 d, PHLPP2 AS ODNsþI/R 5 d group, respectively (scale bar: 5 lm). (C) Quantification data of neuron numbers in CA1 region in the different groups. The data were shown in Mean and SEM with six repeats. nPo0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
trocauterization of the vertebral arteries bilaterally and placement of a traumatic clasps around the common carotid arteries without interruption of the arterial blood flow. On the following day, forebrain ischemia was induced by tightening the clasps for 15 min. Body temperature was maintained at 37 1C during and after occlusion using heat lamps. The rats were awake before ischemia and had normal vital signs.
15 min. The same volume of missense oligonucleotides (MS ODNs) (dissolved in TE buffer) was used as the negative control. The sequences of PHLPP2 antisense oligonucleotide and negative control (Sangon Biotech, Shanghai, China) are as following: AS ODNs (1): 5' TTTCCAAACGGTTTCCTGC 3'; MS ODNs: 5' AAGGCACTTGTTGACGAAC 3'.
4.4. 4.3.
Tissue preparation
Antisense oligonucleotide application
The antisense oligonucleotide was applied as previously demonstrated (Pei et al., 2005). Briefly, the anesthetic rats were placed on a stereotaxic instrument. A parameter (AP: 0.8 mm, ML: 1.5 mm, DV: 3.5 mm) was used for antisense oligonucleotides (AS ODNs) injection into ventricle. AS (10 ml, 10 nM dissolved in TE buffer) were injected for consecutive three days before ischemia (once a day). The injection was completed in 30 min and the needle remained for another
For brain tissue preparation, rats were sacrificed under anesthesia at several time points of reperfusion after 15 min of global cerebral ischemia. Whole brains were removed for dissections and the hippocampal CA1 regions were microdissected from both sides of the hippocampal fissure and immediately frozen in liquid nitrogen. If necessary, cytosol fractions and nuclear fractions were extracted as previously described (Ogita and Yoneda, 1994). Briefly, tissue samples were homogenized in 1.5 ml of 10 mM HEPES, pH 7.9, 0.5 mM MgCl2, 10 Mm KCl,
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USA, 1:200) and β-actin (Santa Cruz, USA, 1:1000) at room temperature for 2 h. Subsequently, the membranes were washed with TBS–T and incubated for 1 h with anti-rabbit or anti-mouse immunoglobulin G (1:10,000 dilutions in TBS– T) at room temperature. After washing three times with TBS– T, the membranes were scanned using an Odyssey Infrared Imaging System (LI-COR, USA). The gray values were analyzed using Image-J software.
4.6.
Fig. 8 – A schematic illustrated the potential pathway underlying I/R injury-induced neuronal death. I/R injury elicited the assembly of FKBP51–PHLPP–AKT signaling complex, which inactivated p-AKT pathway and activated JNKs pathway. The imbalances of the pro-survival and cell death pathways lead to caspase-3 cleavage and neuronal apoptosis.
0.1 mM EDTA, 0.5 mM EGTA, 50 mM NaF, 5 mM DTT, 20 Mm, βphosphoglycerol, 1 mM Na3VO4, 1% NP-40, 1 mM benzamidine and enzyme inhibitors: 1 mM PMSF, 5 μg/ml each of pepstatin A, leupeptin, aprotinin, and were then centrifuged for 15 min at 10,000 g at 4 1C. The supernatant, which corresponded to the cytosolic fraction was collected, immediately and stored at 80 1C until use. The protein concentrations were determined using Bradford method with bovine serum albumin (BSA) as standard.
4.5.
Immunoprecipitation and western blotting
The protein samples (400 μg) were dissolved in 320 μl immunoprecipitation (IP) buffer (with proteases inhibitor), followed by adding 5–6 μl primary antibody (PHLPP2, AKT or FKBP51) and 20 mM DTT (5 μl). The samples were mixed at 4 1C overnight. 20 μl ProteinA/GPLUS–Agarose was added into the sample and mixed again for 2–4 h. After centrifugation (10,000 g, 3 min) at 4 1C, the precipitation was washed three time using IP buffer. After vortexes, the mixture was kept in boiling water for 10 min, and then went through centrifugation. The supernatant was separated using 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose membranes through semi-dry transferring method, which were blocked by 5% non-fat dried milk in Tris-Buffered Saline and Tween 20 (TBS–T) at room temperature for 2 h and incubated with anti-FKBP51 (sc-13983) antibody (Santa Cruz, USA, 1:500), anti-PHLPP2 (sc-137663) antibody (Santa Cruz, USA, 1:100), anti-AKT (sc-8312) antibody (Santa Cruz, USA, 1:500), anti-pAKT (Ser 473) (sc-33437) antibody (Santa Cruz, USA, 1:200), anti-p-JNK (G-7) (sc-6254) antibody (Santa Cruz, USA, 1:200), anti-JNK (D-2) (sc-7345) antibody (Santa Cruz, USA, 1:200), anti- p-c-Jun (Ser63) (54B3) antibody (Cell Signaling, USA, 1:200), anti-c-Jun(B-1) (sc-166540) antibody (Santa Cruz, USA, 1:100), anti-cleaved-casepase-3 antibody (Cell Signaling,
Cresyl violet staining
After anesthesia with intraperitoneal (i.p) injection of 20% chloral hydrate, rats were perfused with 4% paraformaldehyde (PFA) through the thoracic cavity. The whole brain was isolated and post fixed in 4% PFA for 12–24 h. After gradient ethanol dehydration, xylene transparent, and embedding in paraffin, the tissues were sectioned into 5 μm slices. After dewaxing and dehydration, standard Cresyl violet staining was applied. The images were photographed under an Olympus microscope (Japan) and analyzed using PM-CM20 system (Olympus, Japan). The survival neurons in CA1 region were counted.
4.7.
Statistical analysis
Data were analyzed using SPSS software (Version 18.0, SPSS, Chicago, USA). Values were expressed as Means7Standard error of the mean (SEM). Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by multiple comparisons with the LSD post-hoc test. A P value of Po0.05 was considered significantly different.
Acknowledgments This study was supported by the funding of Technology Bureau from Xuzhou (XF10C030, 2010).
r e f e r e nc e s
Abe, J., Baines, C.P., Berk, B.C., 2000. Role of mitogen-activated protein kinases in ischemia and reperfusion injury: the good and the bad. Circ. Res. 86, 607–609. Allen, C.L., Bayraktutan, U., 2008. Risk factors for ischaemic stroke. Int. J. Stroke 3, 105–116. Andreozzi, F., Procopio, C., Greco, A., Mannino, G.C., Miele, C., Raciti, G.A., Iadicicco, C., Beguinot, F., Pontiroli, A.E., Hribal, M.L., Folli, F., Sesti, G., 2011. Increased levels of the Aktspecific phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP)-1 in obese participants are associated with insulin resistance. Diabetologia 54, 1879–1887. Borsello, T., Forloni, G., 2007. JNK signalling: a possible target to prevent neurodegeneration. Curr. Pharm. Des. 13, 1875–1886. Brognard, J., Sierecki, E., Gao, T., Newton, A.C., 2007. PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol. Cell 25, 917–931. Chan, J.H., Lim, S., Wong, W.S., 2006. Antisense oligonucleotides: from design to therapeutic application. Clin. Exp. Pharmacol. Physiol. 33, 533–540.
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Chen, B., Van Winkle, J.A., Lyden, P.D., Brown, J.H., Purcell, N.H., 2013. PHLPP1 gene deletion protects the brain from ischemic injury. J. Cereb. Blood Flow Metab. 33, 196–204. Clemens, J.A., Stephenson, D.T., Yin, T., Smalstig, E.B., Panetta, J.A., Little, S.P., 1998. Drug-induced neuroprotection from global ischemia is associated with prevention of persistent but not transient activation of nuclear factorkappaB in rats. Stroke 29, 677–682. Crotty, T.M., Nakano, T., Stafforini, D.M., Topham, M.K., 2013. Diacylglycerol kinase delta modulates Akt phosphorylation through pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2). J. Biol. Chem. 288, 1439–1447. Datta, S.R., Brunet, A., Greenberg, M.E., 1999. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927. Deb, P., Sharma, S., Hassan, K.M., 2010. Pathophysiologic mechanisms of acute ischemic stroke: an overview with emphasis on therapeutic significance beyond thrombolysis. Pathophysiology 17, 197–218. Fabian, A.K., Marz, A., Neimanis, S., Biondi, R.M., Kozany, C., Hausch, F., 2013. InterAKTions with FKBPs—mutational and pharmacological exploration. PLoS One 8, e57508. Figueroa, C., Tarras, S., Taylor, J., Vojtek, A.B., 2003. Akt2 negatively regulates assembly of the POSH–MLK–JNK signaling complex. J. Biol. Chem. 278, 47922–47927. Gao, T., Furnari, F., Newton, A.C., 2005. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol. Cell 18, 13–24. Goldstein, L.B., Adams, R., Alberts, M.J., Appel, L.J., Brass, L.M., Bushnell, C.D., Culebras, A., DeGraba, T.J., Gorelick, P.B., Guyton, J.R., Hart, R.G., Howard, G., Kelly-Hayes, M., Nixon, J.V., Sacco, R.L., American Heart, A., American Stroke Association Stroke, C., 2006. Primary prevention of ischemic stroke: a guideline from the American Heart Association/ American Stroke Association Stroke Council: cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group. Circulation 113, e873–923. Hui, L., Pei, D.S., Zhang, Q.G., Guan, Q.H., Zhang, G.Y., 2005. The neuroprotection of insulin on ischemic brain injury in rat hippocampus through negative regulation of JNK signaling pathway by PI3K/Akt activation. Brain Res. 1052, 1–9. Jackson, T.C., Verrier, J.D., Semple-Rowland, S., Kumar, A., Foster, T.C., 2010. PHLPP1 splice variants differentially regulate AKT and PKCalpha signaling in hippocampal neurons: characterization of PHLPP proteins in the adult hippocampus. J. Neurochem. 115, 941–955. Jackson, T.C., Verrier, J.D., Drabek, T., Janesko-Feldman, K., Gillespie, D.G., Uray, T., Dezfulian, C., Clark, R.S., Bayir, H., Jackson, E.K., Kochanek, P.M., 2013. Pharmacological inhibition of pleckstrin homology domain leucine-rich repeat protein phosphatase is neuroprotective: differential effects on astrocytes. J. Pharmacol. Exp. Ther. 347, 516–528.
Miyamoto, S., Purcell, N.H., Smith, J.M., Gao, T., Whittaker, R., Huang, K., Castillo, R., Glembotski, C.C., Sussman, M.A., Newton, A.C., Brown, J.H., 2010. PHLPP-1 negatively regulates Akt activity and survival in the heart. Circ. Res. 107, 476–484. Morrison, R.S., Kinoshita, Y., Johnson, M.D., Ghatan, S., Ho, J.T., Garden, G., 2002. Neuronal survival and cell death signaling pathways. Adv. Exp. Med. Biol. 513, 41–86. Ogita, K., Yoneda, Y., 1994. Partial purification of [3H]glutamateassociating-proteins with sensitivity to displacement by N-methyl-D-aspartate from rat brain. Neurochem. Int. 25, 111–121. Pei, D.S., Guan, Q.H., Sun, Y.F., Zhang, Q.X., Xu, T.L., Zhang, G.Y., 2005. Neuroprotective effects of GluR6 antisense oligodeoxynucleotides on transient brain ischemia/ reperfusion-induced neuronal death in rat hippocampal CA1 region. J. Neurosci. Res. 82, 642–649. Pei, H., Li, L., Fridley, B.L., Jenkins, G.D., Kalari, K.R., Lingle, W., Petersen, G., Lou, Z., Wang, L., 2009. FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. Cancer Cell 16, 259–266. Shen, H.M., Liu, Z.G., 2006. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic. Biol. Med. 40, 928–939. Shiojima, I., Walsh, K., 2006. Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev. 20, 3347–3365. Vassalli, G., Milano, G., Moccetti, T., 2012. Role of mitogenactivated protein kinases in myocardial ischemia-reperfusion injury during heart transplantation. J. Transplant. 2012, 928954. Wang, L., 2011. FKBP51 regulation of AKT/protein kinase B phosphorylation. Curr. Opin. Pharmacol. 11, 360–364. Whitmarsh, A.J., Kuan, C.Y., Kennedy, N.J., Kelkar, N., Haydar, T.F., Mordes, J.P., Appel, M., Rossini, A.A., Jones, S.N., Flavell, R.A., Rakic, P., Davis, R.J., 2001. Requirement of the JIP1 scaffold protein for stress-induced JNK activation. Genes Dev. 15, 2421–2432. Woodruff, T.M., Thundyil, J., Tang, S.C., Sobey, C.G., Taylor, S.M., Arumugam, T.V., 2011. Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol. Neurodegener. 6, 11. Xu, N., Lao, Y., Zhang, Y., Gillespie, D.A., 2012. Akt: a double-edged sword in cell proliferation and genome stability. J. Oncol. 2012, 951724. Zhang, Q.G., Wu, D.N., Han, D., Zhang, G.Y., 2007. Critical role of PTEN in the coupling between PI3K/Akt and JNK1/2 signaling in ischemic brain injury. FEBS Lett. 581, 495–505. Zhu, G., Wang, X., Wu, S., Li, Q., 2012. Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against MPPþ-induced human neuroblastoma SH-SY5Y cell death. Neurochem. Int. 60, 400–408. Zhu, J., Shen, W., Gao, L., Gu, H., Shen, S., Wang, Y., Wu, H., Guo, J., 2013. PI3K/Akt-independent negative regulation of JNK signaling by MKP-7 after cerebral ischemia in rat hippocampus. BMC Neurosci. 14, 1.
Available online at www.sciencedirect.com
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Research Report
Assembly of the FKBP51–PHLPP2–AKT signaling complex in cerebral ischemia/reperfusion injury in rats Xiu-E Weia,n, Feng-Yu Zhangb, Kai Wanga, Qing-Xiu Zhanga, Liang-Qun Ronga a
The Second Affiliated Hospital of Xuzhou Medical University, Xuzhou 221000, China Liaocheng Hospital, Liaocheng 252000, China
b
ar t ic l e in f o
abs tra ct
Article history:
The imbalance of cell pro-death and pro-survival signaling pathways determines the neuronal
Accepted 8 April 2014
fate during cerebral ischemia/reperfusion (I/R) injury. However, the biological mechanisms
Available online 16 April 2014
regulating the balance between activation of the pro-death or the pro-survival signaling
Keywords:
pathways remain unclear. In this study, a rat model of I/R injury was established using four-
FKBP51–PHLPP–AKT signaling
vessel occlusion followed by different times of reperfusion. I/R injury did not affect the level of
complex
FK506 binding protein 51 (FKBP51), PH domain and leucine rich repeat protein phosphatases
PHLPP2
(PHLPP)-2, and AKT, but induced assembly of the FKBP51-PHLPP2-AKT signaling complex, as
p-AKT
indicated by the enhancement of interactions among these compounds following reperfusion.
p-JNKs
Using an antisense oligonucleotide, PHLPP2 expression was effectively inhibited. Critically, the
Cell death
inhibition of PHLPP2 prohibited the interactions of FKBP51, PHLPP2 and AKT, reversed the decrease of p-AKT expression and increased the expression of p-JNKs and p-c-Jun elicited by I/R injury. In addition, PHLPP2 inhibition reversed I/R-injury-induced Caspase-3 cleavage and loss of pyramid neurons in the CA1 region of hippocampus. The results of the current study indicate that the assembly of the FKBP51-PHLPP2-AKT signaling complex plays a critical role in mediating cell death in I/R injury. The inhibition of PHLPP2 via antisense oligonucleotide treatment may be an effective method to prohibit the assembly of the FKBP51–PHLPP–AKT signaling complex, thus balancing the cell pro-survival and pro-death signaling pathways ultimately mitigating cell death in I/R injury. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke is one of the most severe cerebrovascular diseases endangering human health. In conjunction with high n
incidence rates, serious sequel, high morbidity and recurrence rates affect the disease therapy accordingly (Goldstein et al., 2006). Therefore, establishing a potential therapeutic target for ischemia/reperfusion (I/R) injury is of high importance. Recently,
Correspondence to:Meijian Road 32, Quanshan District, Xuzhou, Jiangsu, China. Fax: þ86 516 85326115. E-mail address: [email protected] (X.-E. Wei).
http://dx.doi.org/10.1016/j.brainres.2014.04.009 0006-8993/& 2014 Elsevier B.V. All rights reserved.
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the pathophysiological pathways of stroke have become of high importance in the study of cerebrovascular disease (Deb et al., 2010). A line of hypothesis on the occurrence, development and prognosis of stroke have previously been proposed (Woodruff et al., 2011), however the balance between the cell survival and cell death signaling pathways, both of which play critical roles in determining neuron survival (Abe et al., 2000; Vassalli et al., 2012), is yet to be explored. Under pathological conditions, it has been reported that the balance between these two signaling pathways is disrupted, ultimately resulting in neuronal injury or cell death (Morrison et al., 2002). The PH domain and leucine rich repeat protein phosphatases (PHLPP) can specifically dephosphorylate the hydrophobic motif, Ser-473 in AKT, thus partially inactivating the kinase (Gao et al., 2005). There are three major subtypes included in the PHLPP2 family, namely PHLPP1α, PHLPP1β and PHLPP2 all with structural similarity including: a putative Ras association domain; a pleckstrin homology domain; a series of leucine-rich repeats; a PP2C phosphatase domain and a C-terminal PDZ ligand. As PHLPP negatively regulates the pro-cell survival protein kinase B (PKB)/ AKT pathway, this phosphatase has recently attracted attention in several lines of research areas including in tumor progression (Gao et al., 2005), diabetes (Andreozzi et al., 2011) and I/R injury (Jackson et al., 2013; Miyamoto et al., 2010). It has been reported that PHLPP is widely expressed in the cytoplasm, the nuclei and the cell membrane of breast, prostate, ovarian and brain tissue (Gao et al., 2005). Importantly, low expression of PHLPP1 has been reported to have potential protective effects against cardiomyocyte or neuron damage (Miyamoto et al., 2010; Shiojima and Walsh, 2006). The exact biological roles of PHLPP2 in cerebral I/R injury currently remain unknown. Both AKT1 and AKT3 are widely expressed in the brain and play critical roles in lowering
cerebral injury (Xu et al., 2012). In addition, PHLPP2 has been reported to negatively regulate AKT1 and AKT3 dephosphorylation (Brognard et al., 2007). The manner in which PHLPP2 regulates p-AKT and the downstream pathways during cerebral I/R injury remains unknown. The FK506 binding protein 51 (FKBP51) not only enhances the interaction between PHLPP and AKT but also increases PHLPP activity, leading to AKT dephosphorylation (Pei et al., 2009; Wang, 2011). The current study aimed to investigate the hypotheses that I/R injury facilitates the assembly of the FKBP51–PHLPP2–AKT signaling complex thus regulating the pro-cell survival and cell death signaling pathways. Using a rat model of cerebral ischemia injury, the current study successfully demonstrated that the assembly of the FKBP51– PHLPP2–AKT signaling complex plays an essential role in mediating cell death during I/R injury. The inhibition of PHLPP2 through antisense oligonucleotide treatment was an effective method which can inhibit the assembly of the FKBP51-PHLPP2-AKT signaling complex and reverse I/R injury-induced disruption of the balance between the cell death and pro-cell survival signaling pathways. The results of the current study provide new initiatives in terms of new therapeutic treatments for I/R injury.
2.
Results
2.1. I/R injury induced assembly of FKBP51–PHLPP2–AKT signaling complex Consistent with previous studies, western blot analyses confirmed that PHLPP2 and FKBP51 proteins were expressed in rat
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Fig. 1 – Ischemia/reperfusion (I/R) injury did not affect PHLPP2 and FKBP51 expression. (A) Representative blots of PHLPP2 expression after different times of reperfusion. (B) Quantification data of PHLPP2 expression. (C) Representative blots of FKBP51 expression after different times of reperfusion. (D) Quantification data of FKBP51 expression. The data were shown in Mean and SEM with six repeats.
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hippocampal tissue (Brognard et al., 2007; Jackson et al., 2010). In addition, compared with sham control, the protein expression of both PHLPP2 and FKBP51 at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h and 3 days after reperfusion were not significantly different (Fig. 1). The interactions between FKBP51, PHLPP2 and AKT proteins were determined using immunoprecipitation (IP). As shown in Fig. 2, FKBP51, PHLPP2 and AKT proteins were immunoprecipitated together, indicating interactions between these three proteins in rat hippocampal tissue. Critically, the interactions of these three proteins gradually enhanced followed by a decrease with the increase of reperfusion time. A significant difference was observed after 6 h reperfusion compared with the sham control.
2.2. PHLPP2 inhibition reduced the assembly of the FKBP51–PHLPP–AKT signaling complex during I/R injury Three PHLPP2 antisense oligonucleotides (AS ODNs) and a missense oligonucleotides (MS ODNs) were designed to screen the antisense oligonucleotide which can block the PHLPP2 expression. As shown in Fig. 3A and B, AS ODNs (5' TTTCCAAACGGTTTCCTGC 3') significantly inhibited
IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 IP:PHLPP2 IB:Akt IB:β-actin
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2.3. PHLPP2 inhibition increased AKT phosphorylation following I/R injury It has previously been reported that the PKB/AKT pathway is important in the pro-cell survival pathway in neurons (Zhu et al., 2012). In contrast, in the current study, as shown in Fig. 4, I/R (6 h) significantly decreased the levels of p-AKT and did not affect the levels of total AKT. Both Tris–EDTA buffer (TE) and MS ODNs treatment did not influence the levels of p-AKT compared with I/R group. In contrast, AS ODNs treatment significantly increased p-AKT levels compared with MS ODNs treatment. These data indicate that inhibition of PHLPP2 can activate the PKB/AKT pathway most likely through preventing the assembly of the FKBP51–PHLPP2–AKT signaling complex.
2.4. Inhibition of PHLPP2 decreased phosphorylation of JNKs following I/R injury
IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 IP:PHLPP2 IB:Akt
O.D.(Folds vs sham)
PHLPP2 expression to approximately 30% of the control level. The PHLPP2 level in the hippocampus after AS ODNs treatment was significantly reduced compared with that after MS ODNs treatment. The influences of PHLPP2 inhibition on the assembly of the FKBP51–PHLPP2–AKT signaling complex was then investigated after I/R injury (6 h). The MS ODNs was used as the negative control. Compared with the MS ODNs, inhibition of PHLPP2 following AS ODNs treatment significantly reduced the assembly of the FKBP51–PHLPP2–AKT signaling complex as evidenced by the decreased interactions between these three proteins (Fig. 3C and D).
Fig. 2 – I/R injury promoted assembly of FKBP51–PHLPP2– AKT signaling complex. (A) The representative immunoprecipitation blots of FKBP51, PHLPP and AKT after different times of reperfusion. (B) Quantification data showing the interactions among these three proteins were increased with the increase of reperfusion time. The data were shown in Mean and SEM with six repeats. nPo0.05 compared with sham control.
It has been reported that activation of the PKB/AKT pathway can protect against ischemia injury by inhibiting the phosphorylation of JNKs either directly or indirectly (Zhang et al., 2007; Zhu et al., 2013). To address the effects of PHLPP2 inhibition on the JNK pathway, the activation of JNKs and c-Jun were investigated. In the current study, I/R injury did not affect total JNK (Fig. 5A and B) and c-Jun (Fig. 5C and D). The peak increase of p-JNKs, p-c-Jun was observed 6 h after reperfusion. Compared with the sham control, I/R (6 h) significantly increased the levels of p-JNKs. Both TE and MS ODNs treatment did not affect p-JNKs levels compared with the control group. Interestingly, AS ODNs treatment significantly decreased p-JNKs levels compared with MS ODNs treatment. The levels of p-c-Jun 6 h after reperfusion were then investigated. As shown in Fig. 5C and D, compared with sham control, I/R (6 h) significantly increased the level of p-c-Jun. Both TE and MS ODNs treatment did not affect the level of p-c-Jun compared with the control group. The AS ODNs treatment significantly decreased p-c-Jun level compared with the MS ODNs treatment group. In addition, the levels of apoptosis “effector” caspase-3 were determined in the different experimental groups. Compared with sham control, I/R (6 h) significantly increased the level of cleaved-caspase-3 level (Fig. 6). Neither TE nor MS ODNs treatment reversed the level of cleaved-caspase-3 compared with the control group. Treatment with the AS ODNs did however significantly decrease the level of cleavedcaspase-3 compared with MS ODNs treatment.
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IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 IP:PHLPP2 IB:Akt IB:β-actin
IB:PHLPP2 IB:β-actin
IP:FKBP51 IB:Akt IP:FKBP51 IB:PHLPP2 2.0
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Fig. 3 – PHLPP2 inhibition using an antisense oligonucleotide prevented against assembly of FKBP51–PHLPP–AKT signaling complex observed 6 h after reperfusion. (A) Representative blots of PHLPP2 expression after antisense oligonucleotide treatment. (B) Quantification data of PHLPP2 expression after antisense oligonucleotide treatment. (C) The representative immunoprecipitation blots of FKBP51, PHLPP and AKT after PHLPP2 inhibition and I/R injury. (D) Quantification data showed the increased interactions among these three proteins after I/R injury were prevented by PHLPP2 inhibition. The data were shown in Mean and SEM with six repeats. *Po0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
2.5. Inhibition of PHLPP2 prevented neuronal death after I/R injury To demonstrate the effects of the assembly of the FKBP51– PHLPP2–AKT signaling complex on neuronal death after I/R injury, Cresyl violet staining was applied to cells to determine the number of pyramid neurons in the CA1 region. As shown in Fig. 7, the pyramid neurons in the sham control group were closely spaced, but not in I/R group, the MS ODNs and the TE groups. This was ameliorated by AS ODNs treatment. The losses of pyramid neurons were observed in I/R and MS ODNs group. However, the loss of pyramid neurons after I/R was significantly reversed with AS ODNs treatment.
3.
Discussion
The risk factors for ischemic stroke have been largely reported, including old age, high blood pressure, heart disease, smoking
and diabetes (Allen and Bayraktutan, 2008). The mechanisms involved in the initiation of ischemic stroke remain controversial with the widely accepted view being that is the disruption of the balance between neuronal cell survival and cell death responsible for ischemic stroke. Among these pathways, the PI3K/AKT and JNKs pathway are two primary pathways playing opposite roles in the determination of cell fate (Hui et al., 2005). Unfortunately, the upstream pathway mediating PI3K/AKT is not well known. Whilst the inhibition of PHLPP1 has been reported to regulate AKT phosphorylation and can significantly increase cell survival and diminish the infarct size after I/R injury (Chen et al., 2013), the exact roles of PHLPP2 in I/R injury is currently not well documented. In the present study, I/R injury did not affect the level of PHLPP2. Interestingly, in the current study, I/R injury promoted the assembly of the FKBP51– PHLPP2–AKT signaling complex. The immunoprecipitation between FKBP51, PHLPP2 and AKT were increased with the increase of reperfusion time. Supporting these findings is a report implicating that FKBP51 acts as a scaffolding protein for
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Fig. 4 – PHLPP2 inhibition prevented against I/R injuryinduced decrease of p-AKT level. (A) Representative blots of p-AKT expression after antisense oligonucleotide treatment. (B) Quantification data of p-AKT expression after antisense oligonucleotide treatment. The data are shown as Mean7SEM with six repeats. nPo0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
AKT and PHLPP and promotes dephosphorylation of AKT in cancer etiology in response to chemotherapy (Pei et al., 2009). The results of the current study indicate that FKBP51 acted as a scaffold protein regulating AKT and PHLPP2 after cerebral I/R injury. The assembly of the FKBP51–PHLPP2–AKT signaling complex may be responsible for the activation of the cell death pathway and consequently cell death. Identifying the drugs that can block the assembly of this signaling complex may be therapeutically important for the prevention of stroke. As FKBP inhibitors are unlikely to inhibit the AKT– FKBP–PHLPP network (Fabian et al., 2013), PHLPP2 may be the possible therapeutic target protein. Inhibition of specific protein expression using AS ODNs is a useful strategy in different disease treatment (Chan et al., 2006). In the current study, the AS ODNs (5' TTTCCAAACGGTTTCCTGC 3') significantly inhibited the protein expression of PHLPP2 to approximately 30% of control levels and the efficiency rate of inhibition was high (70%). Importantly, this AS ODNs inhibited the assembly of the FKBP51–PHLPP2–AKT signaling complex, which appeared 6 h after reperfusion. After inhibition of PHLPP2, the interaction of FKBP51 with AKT during I/R injury was decreased. It is may be postulated that interaction
between FKBP51 and PHLPP2 was reduced due to the decreased expression of PHLPP2. Surprisingly, with the similar amount of input protein PHLPP2, the immunoprecipitated AKT levels were reduced after AS ODNs treatment following I/R injury. These data suggest that the interaction between PHLPP2 and AKT was decreased most likely through the decreased scaffolding effect of FKBP51. Whilst the direct effect of PHLPP2 on AKT dephosphorylation was not excluded from the current study, unaltered PHLPP2 expression during I/R injury partially explained the important role of the assembly of the FKBP51–PHLPP2–AKT signaling complex, rather than the actual level of PHLPP2 itself following I/R injury. Comparably, β-arrestin 1 has been reported to specifically scaffold PHLPP2 and Akt1 to regulate phosphorylation of Akt (Crotty et al., 2013). That study and ours emphasized the importance of signaling complex in regulating cell prosurvival or pro-death pathways. The inhibition of PHLPP2 using AS ODNs may be an effective way to prevent I/R injury as such inhibition in the current study blocked the assembly of the FKBP51–PHLPP2– AKT signaling complex. To this end, the current study confirmed that PHLPP2 inhibition induces blockage of the assembly of the FKBP51–PHLPP–AKT signaling complex thus contributing to the promotion of neuronal cell survival or blockage of neuron death. The PI3K/AKT pathway is an important pro-cell survival pathway in mammalian cells under different pathological conditions (Datta et al., 1999; Zhu et al., 2012). The blockage of this pathway can lead to cell death, whereas activation of this pathway prevents cell death. Consistently, in this study, the level of p-AKT was significantly decreased, indicating that the PI3K/AKT pathway was inhibited after I/R injury. Knockdown of FKBP1 or the TRP domain has been reported to attenuate the interaction between AKT and PHLPP (Pei et al., 2009). The inhibition using the PHLPP2 AS ODNs further supports this notion. In the current study, PHLPP2 inhibition compromised the interactions of these three proteins and in turn, the level of p-AKT was enhanced in the experimental group compared with I/R group. These data indicate that p-AKT level is possibly regulated by the FKBP51–PHLPP2–AKT signaling complex. The present study therefore clarifies a novel mediating mechanism of PHLPP2 on p-AKT after I/R injury. Unlike the PI3K/AKT pathway, the JNKs pathway plays an opposing role in the mediation of cell death (Shen and Liu, 2006). The activation of the JNKs pathway can indeed exacerbate neurodegeneration (Borsello and Forloni, 2007). A scaffold protein, c-Jun NH2 terminal kinase interacting protein-1 (JIP-1) can activate JNK through direct interaction with JNK (Whitmarsh et al., 2001). However, interaction of AKT with JIP-1 compromises the interaction of JIP-1 with JNK, in turn inhibiting the activation of the JNKs pathway and antagonizing neuron apoptosis (Whitmarsh et al., 2001). Prior research has shown that AKT combined with POSH/MLK/MKK/JNK forms a mediating module, which can inhibit the activation of the JNK pathway (Figueroa et al., 2003). In the current study, I/R injury activated the JNKs pathway, as indicated by increased levels of p-JNKs and increased phosphorylation of the nuclear substrate c-Jun. Concurrently, inhibition of PHLPP2 using the AS ODNs could inactivate the JNKs pathway. To further confirm the injury of I/R and the protection provided by PHLPP2 inhibition, the cleaved
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4.2.
Animals and model
Male Sprague-Dawley rats (weighing 220–250 g) were purchased from the Experimental Animals Center of Xuzhou Medical College (China). Transient forebrain ischemia was induced by four-vessel occlusion (4-VO) as previously described (Clemens et al., 1998). Briefly, rats were prepared for forebrain ischemia under 20% chloral hydrate (300–350 mg/kg) anesthesia by elec-
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The animal care and surgical procedures were performed under the established standards of the Second Affiliated Hospital of Xuzhou Medical College (China).
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O.D.(Cleaved-Caspase-3/β -actin)
caspase-3 expression and cell loss in the CA1 region were determined. As expected, I/R injury-induced apoptosis pathway was inhibited by PHLPP2 inhibition as indicated by decreased cleaved caspase-3 level. In conclusion, the current study demonstrates that I/R injury does not affect FKBP51, PHLPP2 and AKT protein expression, but increases the assembly of the FKBP51–PHLPP2–AKT signaling complex (Fig. 8). PHLPP2 inhibition using a specific antisense oligonucleotide could effectively compromise assembly of the FKBP51–PHLPP2–AKT signaling complex, which most likely activates p-AKT and blocks the JNKs pathway, leading to neuronal protection. The current study provides a novel stratagem for stroke prevention and therapy.
Fig. 6 – PHLPP2 inhibition prevented against I/R injury-induced caspase-3 cleavage. (A) Representative blots of cleaved-caspase3 expression after antisense oligonucleotide treatment. (B) Quantification data of cleaved-caspase-3 expression after antisense oligonucleotide treatment. The data were shown in Mean and SEM with six repeats. *Po0.05 compared with sham control. #Po0.05 compared with missense oligonucleotides treatment.
IB:JNK
IB:c-Jun
IB:p-JNK
IB:p-c-Jun
IB:β-actin
IB:β-actin
Fig. 5 – PHLPP2 inhibition prevented against I/R injury-induced activation of JNKs pathway. (A) Representative blots of p-JNK expression after antisense oligonucleotide treatment. (B) Quantification data of p-JNK expression after antisense oligonucleotide treatment. (C) Representative blots of p-c-Jun expression after antisense oligonucleotide treatment. (D) Quantification data of p-c-Jun expression after antisense oligonucleotide treatment. The data were shown in Mean and SEM with six repeats. *Po0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
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AS ODN
MS ODN
Neurons /mm length of CA1
TE
I/R 3 d
Sham
brain research 1566 (2014) 60–68
Fig. 7 – PHLPP2 inhibition prevented I/R injury-induced cell loss in CA1 region. (A) Representative image of hippocampus after Cresyl violet staining (scale bar: 100 lm). The black rectangle indicates the CA1 region. (B) The representative images of CA1 region in sham control, I/R 5d, TEþI/R 5 d, PHLPP2 MS ODNsþI/R 5 d, PHLPP2 AS ODNsþI/R 5 d group, respectively (scale bar: 5 lm). (C) Quantification data of neuron numbers in CA1 region in the different groups. The data were shown in Mean and SEM with six repeats. nPo0.05 compared with sham control. #Po0.05 compared with missense oligonucleotide treatment.
trocauterization of the vertebral arteries bilaterally and placement of a traumatic clasps around the common carotid arteries without interruption of the arterial blood flow. On the following day, forebrain ischemia was induced by tightening the clasps for 15 min. Body temperature was maintained at 37 1C during and after occlusion using heat lamps. The rats were awake before ischemia and had normal vital signs.
15 min. The same volume of missense oligonucleotides (MS ODNs) (dissolved in TE buffer) was used as the negative control. The sequences of PHLPP2 antisense oligonucleotide and negative control (Sangon Biotech, Shanghai, China) are as following: AS ODNs (1): 5' TTTCCAAACGGTTTCCTGC 3'; MS ODNs: 5' AAGGCACTTGTTGACGAAC 3'.
4.4. 4.3.
Tissue preparation
Antisense oligonucleotide application
The antisense oligonucleotide was applied as previously demonstrated (Pei et al., 2005). Briefly, the anesthetic rats were placed on a stereotaxic instrument. A parameter (AP: 0.8 mm, ML: 1.5 mm, DV: 3.5 mm) was used for antisense oligonucleotides (AS ODNs) injection into ventricle. AS (10 ml, 10 nM dissolved in TE buffer) were injected for consecutive three days before ischemia (once a day). The injection was completed in 30 min and the needle remained for another
For brain tissue preparation, rats were sacrificed under anesthesia at several time points of reperfusion after 15 min of global cerebral ischemia. Whole brains were removed for dissections and the hippocampal CA1 regions were microdissected from both sides of the hippocampal fissure and immediately frozen in liquid nitrogen. If necessary, cytosol fractions and nuclear fractions were extracted as previously described (Ogita and Yoneda, 1994). Briefly, tissue samples were homogenized in 1.5 ml of 10 mM HEPES, pH 7.9, 0.5 mM MgCl2, 10 Mm KCl,
brain research 1566 (2014) 60–68
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USA, 1:200) and β-actin (Santa Cruz, USA, 1:1000) at room temperature for 2 h. Subsequently, the membranes were washed with TBS–T and incubated for 1 h with anti-rabbit or anti-mouse immunoglobulin G (1:10,000 dilutions in TBS– T) at room temperature. After washing three times with TBS– T, the membranes were scanned using an Odyssey Infrared Imaging System (LI-COR, USA). The gray values were analyzed using Image-J software.
4.6.
Fig. 8 – A schematic illustrated the potential pathway underlying I/R injury-induced neuronal death. I/R injury elicited the assembly of FKBP51–PHLPP–AKT signaling complex, which inactivated p-AKT pathway and activated JNKs pathway. The imbalances of the pro-survival and cell death pathways lead to caspase-3 cleavage and neuronal apoptosis.
0.1 mM EDTA, 0.5 mM EGTA, 50 mM NaF, 5 mM DTT, 20 Mm, βphosphoglycerol, 1 mM Na3VO4, 1% NP-40, 1 mM benzamidine and enzyme inhibitors: 1 mM PMSF, 5 μg/ml each of pepstatin A, leupeptin, aprotinin, and were then centrifuged for 15 min at 10,000 g at 4 1C. The supernatant, which corresponded to the cytosolic fraction was collected, immediately and stored at 80 1C until use. The protein concentrations were determined using Bradford method with bovine serum albumin (BSA) as standard.
4.5.
Immunoprecipitation and western blotting
The protein samples (400 μg) were dissolved in 320 μl immunoprecipitation (IP) buffer (with proteases inhibitor), followed by adding 5–6 μl primary antibody (PHLPP2, AKT or FKBP51) and 20 mM DTT (5 μl). The samples were mixed at 4 1C overnight. 20 μl ProteinA/GPLUS–Agarose was added into the sample and mixed again for 2–4 h. After centrifugation (10,000 g, 3 min) at 4 1C, the precipitation was washed three time using IP buffer. After vortexes, the mixture was kept in boiling water for 10 min, and then went through centrifugation. The supernatant was separated using 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose membranes through semi-dry transferring method, which were blocked by 5% non-fat dried milk in Tris-Buffered Saline and Tween 20 (TBS–T) at room temperature for 2 h and incubated with anti-FKBP51 (sc-13983) antibody (Santa Cruz, USA, 1:500), anti-PHLPP2 (sc-137663) antibody (Santa Cruz, USA, 1:100), anti-AKT (sc-8312) antibody (Santa Cruz, USA, 1:500), anti-pAKT (Ser 473) (sc-33437) antibody (Santa Cruz, USA, 1:200), anti-p-JNK (G-7) (sc-6254) antibody (Santa Cruz, USA, 1:200), anti-JNK (D-2) (sc-7345) antibody (Santa Cruz, USA, 1:200), anti- p-c-Jun (Ser63) (54B3) antibody (Cell Signaling, USA, 1:200), anti-c-Jun(B-1) (sc-166540) antibody (Santa Cruz, USA, 1:100), anti-cleaved-casepase-3 antibody (Cell Signaling,
Cresyl violet staining
After anesthesia with intraperitoneal (i.p) injection of 20% chloral hydrate, rats were perfused with 4% paraformaldehyde (PFA) through the thoracic cavity. The whole brain was isolated and post fixed in 4% PFA for 12–24 h. After gradient ethanol dehydration, xylene transparent, and embedding in paraffin, the tissues were sectioned into 5 μm slices. After dewaxing and dehydration, standard Cresyl violet staining was applied. The images were photographed under an Olympus microscope (Japan) and analyzed using PM-CM20 system (Olympus, Japan). The survival neurons in CA1 region were counted.
4.7.
Statistical analysis
Data were analyzed using SPSS software (Version 18.0, SPSS, Chicago, USA). Values were expressed as Means7Standard error of the mean (SEM). Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by multiple comparisons with the LSD post-hoc test. A P value of Po0.05 was considered significantly different.
Acknowledgments This study was supported by the funding of Technology Bureau from Xuzhou (XF10C030, 2010).
r e f e r e nc e s
Abe, J., Baines, C.P., Berk, B.C., 2000. Role of mitogen-activated protein kinases in ischemia and reperfusion injury: the good and the bad. Circ. Res. 86, 607–609. Allen, C.L., Bayraktutan, U., 2008. Risk factors for ischaemic stroke. Int. J. Stroke 3, 105–116. Andreozzi, F., Procopio, C., Greco, A., Mannino, G.C., Miele, C., Raciti, G.A., Iadicicco, C., Beguinot, F., Pontiroli, A.E., Hribal, M.L., Folli, F., Sesti, G., 2011. Increased levels of the Aktspecific phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP)-1 in obese participants are associated with insulin resistance. Diabetologia 54, 1879–1887. Borsello, T., Forloni, G., 2007. JNK signalling: a possible target to prevent neurodegeneration. Curr. Pharm. Des. 13, 1875–1886. Brognard, J., Sierecki, E., Gao, T., Newton, A.C., 2007. PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol. Cell 25, 917–931. Chan, J.H., Lim, S., Wong, W.S., 2006. Antisense oligonucleotides: from design to therapeutic application. Clin. Exp. Pharmacol. Physiol. 33, 533–540.
68
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Chen, B., Van Winkle, J.A., Lyden, P.D., Brown, J.H., Purcell, N.H., 2013. PHLPP1 gene deletion protects the brain from ischemic injury. J. Cereb. Blood Flow Metab. 33, 196–204. Clemens, J.A., Stephenson, D.T., Yin, T., Smalstig, E.B., Panetta, J.A., Little, S.P., 1998. Drug-induced neuroprotection from global ischemia is associated with prevention of persistent but not transient activation of nuclear factorkappaB in rats. Stroke 29, 677–682. Crotty, T.M., Nakano, T., Stafforini, D.M., Topham, M.K., 2013. Diacylglycerol kinase delta modulates Akt phosphorylation through pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2). J. Biol. Chem. 288, 1439–1447. Datta, S.R., Brunet, A., Greenberg, M.E., 1999. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927. Deb, P., Sharma, S., Hassan, K.M., 2010. Pathophysiologic mechanisms of acute ischemic stroke: an overview with emphasis on therapeutic significance beyond thrombolysis. Pathophysiology 17, 197–218. Fabian, A.K., Marz, A., Neimanis, S., Biondi, R.M., Kozany, C., Hausch, F., 2013. InterAKTions with FKBPs—mutational and pharmacological exploration. PLoS One 8, e57508. Figueroa, C., Tarras, S., Taylor, J., Vojtek, A.B., 2003. Akt2 negatively regulates assembly of the POSH–MLK–JNK signaling complex. J. Biol. Chem. 278, 47922–47927. Gao, T., Furnari, F., Newton, A.C., 2005. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol. Cell 18, 13–24. Goldstein, L.B., Adams, R., Alberts, M.J., Appel, L.J., Brass, L.M., Bushnell, C.D., Culebras, A., DeGraba, T.J., Gorelick, P.B., Guyton, J.R., Hart, R.G., Howard, G., Kelly-Hayes, M., Nixon, J.V., Sacco, R.L., American Heart, A., American Stroke Association Stroke, C., 2006. Primary prevention of ischemic stroke: a guideline from the American Heart Association/ American Stroke Association Stroke Council: cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group. Circulation 113, e873–923. Hui, L., Pei, D.S., Zhang, Q.G., Guan, Q.H., Zhang, G.Y., 2005. The neuroprotection of insulin on ischemic brain injury in rat hippocampus through negative regulation of JNK signaling pathway by PI3K/Akt activation. Brain Res. 1052, 1–9. Jackson, T.C., Verrier, J.D., Semple-Rowland, S., Kumar, A., Foster, T.C., 2010. PHLPP1 splice variants differentially regulate AKT and PKCalpha signaling in hippocampal neurons: characterization of PHLPP proteins in the adult hippocampus. J. Neurochem. 115, 941–955. Jackson, T.C., Verrier, J.D., Drabek, T., Janesko-Feldman, K., Gillespie, D.G., Uray, T., Dezfulian, C., Clark, R.S., Bayir, H., Jackson, E.K., Kochanek, P.M., 2013. Pharmacological inhibition of pleckstrin homology domain leucine-rich repeat protein phosphatase is neuroprotective: differential effects on astrocytes. J. Pharmacol. Exp. Ther. 347, 516–528.
Miyamoto, S., Purcell, N.H., Smith, J.M., Gao, T., Whittaker, R., Huang, K., Castillo, R., Glembotski, C.C., Sussman, M.A., Newton, A.C., Brown, J.H., 2010. PHLPP-1 negatively regulates Akt activity and survival in the heart. Circ. Res. 107, 476–484. Morrison, R.S., Kinoshita, Y., Johnson, M.D., Ghatan, S., Ho, J.T., Garden, G., 2002. Neuronal survival and cell death signaling pathways. Adv. Exp. Med. Biol. 513, 41–86. Ogita, K., Yoneda, Y., 1994. Partial purification of [3H]glutamateassociating-proteins with sensitivity to displacement by N-methyl-D-aspartate from rat brain. Neurochem. Int. 25, 111–121. Pei, D.S., Guan, Q.H., Sun, Y.F., Zhang, Q.X., Xu, T.L., Zhang, G.Y., 2005. Neuroprotective effects of GluR6 antisense oligodeoxynucleotides on transient brain ischemia/ reperfusion-induced neuronal death in rat hippocampal CA1 region. J. Neurosci. Res. 82, 642–649. Pei, H., Li, L., Fridley, B.L., Jenkins, G.D., Kalari, K.R., Lingle, W., Petersen, G., Lou, Z., Wang, L., 2009. FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. Cancer Cell 16, 259–266. Shen, H.M., Liu, Z.G., 2006. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic. Biol. Med. 40, 928–939. Shiojima, I., Walsh, K., 2006. Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev. 20, 3347–3365. Vassalli, G., Milano, G., Moccetti, T., 2012. Role of mitogenactivated protein kinases in myocardial ischemia-reperfusion injury during heart transplantation. J. Transplant. 2012, 928954. Wang, L., 2011. FKBP51 regulation of AKT/protein kinase B phosphorylation. Curr. Opin. Pharmacol. 11, 360–364. Whitmarsh, A.J., Kuan, C.Y., Kennedy, N.J., Kelkar, N., Haydar, T.F., Mordes, J.P., Appel, M., Rossini, A.A., Jones, S.N., Flavell, R.A., Rakic, P., Davis, R.J., 2001. Requirement of the JIP1 scaffold protein for stress-induced JNK activation. Genes Dev. 15, 2421–2432. Woodruff, T.M., Thundyil, J., Tang, S.C., Sobey, C.G., Taylor, S.M., Arumugam, T.V., 2011. Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol. Neurodegener. 6, 11. Xu, N., Lao, Y., Zhang, Y., Gillespie, D.A., 2012. Akt: a double-edged sword in cell proliferation and genome stability. J. Oncol. 2012, 951724. Zhang, Q.G., Wu, D.N., Han, D., Zhang, G.Y., 2007. Critical role of PTEN in the coupling between PI3K/Akt and JNK1/2 signaling in ischemic brain injury. FEBS Lett. 581, 495–505. Zhu, G., Wang, X., Wu, S., Li, Q., 2012. Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against MPPþ-induced human neuroblastoma SH-SY5Y cell death. Neurochem. Int. 60, 400–408. Zhu, J., Shen, W., Gao, L., Gu, H., Shen, S., Wang, Y., Wu, H., Guo, J., 2013. PI3K/Akt-independent negative regulation of JNK signaling by MKP-7 after cerebral ischemia in rat hippocampus. BMC Neurosci. 14, 1.
