Heat shock protein 72 inhibits c-Jun N-terminal kinase 3 signaling pathway via Akt1 during cerebral ischemia

Journal of the Neurological Sciences 317 (2012) 123–129

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Heat shock protein 72 inhibits c-Jun N-terminal kinase 3 signaling pathway via Akt1 during cerebral ischemia Dashi Qi a, 1, Hongzhi Liu a, 1, Jian Niu b, Xing Fan c, Xiangru Wen a, Yang Du a, Jie Mou d, Dongsheng Pei e, Zhian Liu f, Zhimin Zong c, Xianyong Wei c, Yuanjian Song a, c, f,⁎ a

Department of Neurobiology, Xuzhou Medical College, China General Surgery of the Hospital Affiliated Xuzhou Medical College, China School of Chemical Engineering, China University of Mining and Technology, China d School of Pharmacy, Xuzhou Medical College, China e Laboratory of Biological Cancer Therapy, Xuzhou Medical College, China f Key Laboratory of Brain Diseases Bioinformation, Xuzhou Medical College, China b c

a r t i c l e

i n f o

Article history: Received 27 November 2011 Received in revised form 20 January 2012 Accepted 10 February 2012 Available online 3 March 2012 Keywords: Cerebral ischemia Heat shock protein 72 Protein kinase B c-Jun N-terminal kinase 3

a b s t r a c t Although recent researches show that Heat Shock Protein 72 (HSP72) plays an important role in neuronal survival, little knowledge is known about the precise mechanisms during cerebral ischemia/reperfusion (I/R). Our present study investigated the neuroprotective mechanisms of HSP72 against ischemic brain injury induced by cerebral I/R. Mild heat shock pretreatment was employed to induce the overexpression of HSP72 by immersing rats into the water bath at 42 °C for 20 min before cerebral I/R. HSP72 antisense oligodeoxynucleotides (ODNs) were used to inhibit HSP72 expression by intracerebroventricular infusion once per day for 3 days before cerebral I/R animal model was induced by four-vessel occlusion for 15 min transient ischemia and then reperfused for various time in Sprague–Dawley rats. Immunoprecipitation and immunoblotting were used to detect the expression of the related proteins. HE-staining and TUNEL-staining were carried out to examine the neuronal death of hippocampal CA1 region. Results showed that mild heat shock could increase the phosphorylation of protein kinase B (Akt), inhibit the assembly of MLK3–MKK7–JNK3 signaling module, diminish the phosphorylation of JNK3 and c-Jun, and decrease the activation of caspase-3. Furthermore, mild heat shock could significantly protect neurons against cerebral I/R. Whereas, all of the aforementioned effects of mild heat shock were reversed by HSP72 antisense ODNs. In summary, our results imply that Akt1 activation is involved in the neuroprotection of HSP72 against ischemic brain injury via suppressing JNK3 signaling pathway and provide a new experimental foundation for stroke therapy. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction It is well known that severe heat shock can cause cell death displaying morphological characteristics of apoptosis, while pretreatment with moderate heat shock is known as protecting cells from stressful treatments, such as ethanol, UV irradiation, doxorubicin (Adriamycin), and tumor necrosis factor (TNF) [1–5]. This protection mainly attributes to members of the HSP70 family, especially 72 kDa heat shock protein (HSP72). HSP72, the major inducible member of the heat shock protein 70 family, has been found protecting cells from certain apoptotic stimuli such as oxidative stress, hypoxia and inflammation [6–10]. Under normal physiological conditions, HSP72 shows very low expression in brain, but it is induced after certain insults such as ischemia or kainic acid-induced excitotoxicity [11,12]. ⁎ Corresponding author at: Department of Neurobiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, Jiangsu, 221002, China. Tel.: + 86 516 83262127. E-mail address: [email protected] (Y. Song). 1 These authors contributed equally to this work.

Although the protective effects of HSP72 against brain injury were discovered several years ago, little knowledge is known about the precise mechanisms during cerebral I/R. Recent studies have demonstrated that ischemic stroke resulting from a transient or permanent reduction in brain blood flow may involve in the regulation of multiple survival and death-signaling pathway which lead to several pathophysiological changes [13]. Phosphatidylinositol 3-kinase (PI3K), which has been extensively studied recently [14], is a key antiapoptotic effector in the growth factor signaling pathway. A variety of stimulation, including tyrosine kinase growth factor receptors, protein phosphatase inhibitors, and stress could activate PI3K, which leads to Akt activation. Akt, a 57 kDa protein-serine/threonine kinase, is a downstream signaling molecule of PI3K [15,16]. Three genes encoding Akt (including akt1, akt2 and akt3) have been identified in mammalian genomes [17]. Akt1 and Akt2 are widely expressed in brain [18,19]. Akt1 activation is correlated with phosphorylation of Thr-308 at its catalytic domain and of Ser473 at the C terminus [20,21]. Furthermore, previous studies have shown that Akt1 phosphorylation at Ser473 plays an important role in neuronal protection

0022-510X/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2012.02.011

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[15]. On the other hand, several targets of Akt1 have been identified to play important roles in the regulation of apoptosis, such as the proapoptotic proteins Bcl-2/Bcl-XL-associated death protein (BAD), caspase-9 and c-Jun N-terminal kinase-3 (JNK3) [22]. JNKs, members of MAPK family, are extremely important in the process of neuronal death [23]. JNKs are encoded by three genes: Jnk1, Jnk2 and Jnk3. Studies show that JNKs have relationship with apoptosis. For example, overexpression of a constitutively activated JNK potentiates apoptosis of PC12 cell in response to nerve growth factor (NGF) deprivation [24]. In addition, JNKs can be activated by a variety of stimuli including ischemia, neurotoxic insults, environmental stress and apoptotic agents [25,26]. Some researches have demonstrated that HSP72 overexpression has relationship with activation of Akt during myocardial I/R injury and doxorubicin-induced heart failure [27,28]. Previous studies also showed that cerebral I/R could inhibit Akt signaling pathway and facilitate the assembly of mixed lineage kinase 3(MLK3)-mitogen-activated kinase kinase 7(MKK7)-JNK3, which further induces the activation of JNK3 leading to neuronal death of hippocampus [29]. However, little knowledge is known about the involvement of Akt1 in hyperthermia-induced HSP72 overexpression in the brain. In this study, we hypothesized that the overexpression of HSP72 induced by mild heat shock can protect neurons against cerebral I/R injury by upregulating the phosphorylation of Akt1(ser473) to suppress the activation of JNK3 and the downstream pro-apoptosis signaling molecule. 2. Materials and methods 2.1. Antibody and reagents Mouse monoclonal anti-HSP72 (SPA-810) was purchased from StressGen Biotechnologies Corp. Mouse monoclonal anti-pAkt1(ser473) (MA1-41002) was obtained from Affinity BioReagents. Mouse monoclonal anti-p-JNKs (sc-6254), rabbit polyclonal antiMLK3 (sc-13072), anti-p-c-Jun (sc-16312-R) and anti-c-Jun (sc-1694) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-MKK7 (#4172), rabbit polyclonal anti-p-MKK7 (#4171) and Rabbit polyclonal anti-p-MLK3 (#2811) were obtained from Cell Signaling Biotechnology. 2.2. Animal surgical procedures Adult male Sprague–Dawley rats weighing 200–250 g (Shanghai Experimental Animal Center, Chinese Academy of Science, Shanghai, China) were given free access to food and water before surgery. All rats were divided into the following groups: sham group, I/R group, I/R and heat shock-treated group, I/R and heat shock-treated along with HSP72-AON-treated group, and I/R and heat shock-treated along with HSP72-NON-treated group. Every group was derived from 6 independent animals. The surgical procedures were approved by the Shanghai Experimental Animal Center. Surgical procedures were conducted under guidelines and the terms of all relevant local legislations. Our best efforts were made to minimize the number of animals used and suffering they got. Transient cerebral ischemia (15 min) was induced by four-vessel occlusion (4-VO) as described before [30]. Briefly, after being anesthetized with chloral hydrate (350 mg/kg, i.p.), rats' both vertebral arteries were occluded permanently by electrocautery and common carotid arteries were exposed. After that, rats were allowed to recover for 24 h and fasted overnight. In order to induce cerebral ischemia, aneurysm clips were used to occlude both carotid arteries. After 15 min of the occlusion, the aneurysm clips were removed for reperfusion. Sham controls were performed using the same surgical procedures, except occluding the carotid arteries. Before inducing transient cerebral ischemia (15 min), transient cerebral ischemia (15 min) was induced by 4-VO. Each rat was anesthetized

with pentobarbital (350 mg/kg, i.p.) and immersed in a water bath at 42 °C (hyperthermia, HT) for 20 min [31]. Rectal temperature was recorded throughout the thermo treatment to monitor changes in body temperature.

2.3. Administration of drugs One hundred micrograms of specific antisense oligonucleotides against HSP72 in 10 μl TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) was administrated to the rats every 24 h for 3 days through brain ventricular injection (anteroposterior, 0.8 mm; lateral, 1.5 mm; depth, 3.5 mm from bregma). The same dose of nonsense oligonucleotides and vehicle was used as control. The sequences of antisense and nonsense are shown as follows: AON: 5′-CACCTTGCCGTGCTGGAA-3′; and NON: 5′-TGGATCCGACATGTCAGA-3′ [32].

2.4. Tissue preparation After reperfusion under anesthesia, rats were decapitated immediately and then the hippocampus were removed and quickly frozen in liquid nitrogen. The hippocampus were homogenized in 1:10 (w/v) ice-cold homogenization buffer containing 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS; Sigma, St. Louis, MO; pH 7.4), 100 mM KCl, 320 mM sucrose, 50 mM NaF, 0.5 mM MgCl2, 0.2 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4 (Sigma), 20 mM sodium pyrophosphate, 20 mM β-phosphogrycerol, 1 mM p-nitrophenyl phosphate (PNPP), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 μg/ml each of leupeptin, aprotinin, pepstatin A. After that, they were centrifuged at 12,000 g for 15 min at 4 °C. Supernatants such as nuclear parts were collected and protein concentrations were determined by the method of Lowry. Samples were stored at −80 °C and were thawed only once.

2.5. Immunoprecipitation Tissue homogenates (400 μg of protein) were diluted fourfold with 50 mM HEPES buffer, pH 7.4, containing 10% glycerol, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, as well as 1 mM of EDTA, EGTA, PMSF, and Na3VO4. Samples were preincubated for 1 h with 20 μl protein A sepharose CL-4B (Amersham, Uppsala, Sweden) at 4 °C, then centrifuged to remove proteins adhering nonspecifically to protein A. The supernatants were incubated with 2 μg primary antibodies for 4 h or overnight at 4 °C. Protein A was added to the tube for another 2 h of incubation. Samples were centrifuged at 10,000 g for 2 min at 4 °C, and the pellets were washed with immunoprecipitation buffer for three times. Bound proteins were eluted by boiling at 100 °C for 5 min in SDS-PAGE loading buffer and then isolated by centrifugation. The supernatants were used for immunoblot analysis.

2.6. Immunoblotting Proteins were separated on polyacrylamide gels and then electrotransferd onto a nitrocellulose membrane (Amersham, Buckinghamshire, United Kingdom). After being blocked for 3 h in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% bovine serum albumin (BSA), membranes were incubated overnight at 4 °C with primary antibodies in TBST containing 1% BSA. Membranes were then washed and incubated with alkaline phosphatase-conjugated secondary antibodies in TBST for 2 h and developed with NBT/BCIP color substrate (Promega, Madison, WI). The densities of the bands on the membrane were scanned and analyzed using an image analyzer (LabWorks Software, Upland, CA).

D. Qi et al. / Journal of the Neurological Sciences 317 (2012) 123–129

2.7. Hematoxylin–eosin staining (HE-staining)

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The rats subjected to 5 days of reperfusion were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffin-embedded brain sections (5 μm) were prepared and stained with hematoxylin and eosin. Histological evaluations were performed with HE-staining for assessment of neuronal damage in the hippocampus. An initial dissector frame was positioned randomly in hippocampal sector and cells in every 10th section throughout the entire hippocampus. The cell numbers in hippocampus were assessed by means of previously published unbiased stereological techniques. In brief, cell counts were performed at ×400 magnification with the use of an Olympus BH-2 microscope connected to a Sony charge-coupled device video camera, a motorize stage system, and commercial stereology software.

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2.8. TUNEL-staining TUNEL-staining was performed using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer's protocol with minor modifications. The paraffin-embedded coronal sections were deparaffinized and rehydrated, and then treated with protease K (20 μg/ml) for 15 min at room temperature. Sections were incubated with reaction buffer containing TdT enzyme at 37 °C for 1 h. After washing with stop/wash buffer, sections were treated with antidigoxigenin conjugate for 30 min at room temperature and subsequently developed color in NBT/BCIP substrate. The TUNEL-positive cell numbers were counted by means of the unbiased stereological methods which are listed in Section 2.7.

Six animals were independently selected as samples in all groups for immunoblotting and histology assays. Image J (Version 1.30 v) analysis software was used for Semiquantitative analysis of the bands. Values were expressed as the means ± SD. Statistical analysis of the results was carried out by one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test. P-values b0.05 were considered significant. 3. Results

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To explore whether HSP72 overexpression can play neuroprotective role against I/R injury, we investigated the effect of heat shock on the survival of CA1 pyramidal neurons in rat hippocampus, where neurons were particularly vulnerable to ischemic injury, at 5 days of reperfusion. HE-staining was used to examine the survival neurons after 5 days reperfusion following 15 min ischemia. The shrunken cells with pyknotic nuclei after ischemia were counted as dead cells. As shown in Fig. 1, transient cerebral ischemia followed by 5 days of reperfusion induced severe cell death. But pretreatment with heat shock for 15 min before ischemia significantly decreased neuronal degeneration. Administration of specific antisense oligonucleotides of HSP72 before heat shock followed by cerebral ischemia aggravated cell death compared to heat shock pretreatment only. As a control, nonsense oligonucleotides had no any effect on cell death during cerebral I/R in the hippocampal CA1 region. TUNEL-staining was used to assess the apoptotic like neurons in the hippocampal CA1 region using the aforementioned treatments in HEstaining at 5 days reperfusion after 15 min ischemia. As shown in Fig. 1, a significant number of TUNEL-positive cells were observed in 5 days after I/R and HSP72 antisense oligodeoxynucleotides (HSP72AON)-treated groups. Heat shock-treated and heat shock-treated together with HSP72 nonsense oligodeoxynucleotides (HSP72-NON)-

Reperfusionfor 5 d Fig. 1. Representative hippocampal photomicrographs of TUNEL-staining and HE-staining. Rats were divided to sham group, 15 min of ischemia followed by 5 day reperfusion group, heat shock (HS) group, HSP72-AON group, and HSP72-NON group (A). The numbers of HE-positive neurons and TUNEL-positive neurons were quantitatively analysised (B). Data were expressed as mean ± SD derived from six independent animals in each experiment group. ⁎P b 0.05 vs. sham group, #P b 0.05 vs. I/R group.

treated markedly decreased the neuronal loss. The results show that HSP72 is capable of protecting neurons against brain ischemic injury. 3.2. Effects of HSP72 on the phosphorylation of Akt1(ser473) In order to confirm the effects of heat shock, HSP72-AON and HSP72-NON on HSP72, we examined the expression of HSP72 after the above preconditions. Fig. 2 illustrated that heat shock significantly

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increased the expression of HSP72, while HSP72-AON decreased this effect, HSP72-NON had no effect on this effect. Previous studies indicated that activation of Akt1(ser473) in the hippocampal CA1 subfield was inhibited to ravine at 0 min and 3 days of reperfusion following 15 min of ischemia [22]. In order to explore the possible role of Akt1 pathway mediated by HSP72, we examined changes of Akt1(ser473) phosphorylation at 0 and 3 days after I/R in both heat shock preconditioned and unpreconditioned hippcoampal CA1 regions. As shown in Fig. 3, heat shock strengthened Akt1(ser473) phosphorylation at 0 min and 3 days of reperfusion compared with that in the I/R control groups, whereas HSP72AON blocked the increase of Akt1(ser473) phosphorylation induced by pretreatment with heat shock only. These data conclusively suggested that HSP72 could increase the phosphorylation of Akt1(ser473) during the process of cerebral I/R. 3.3. Effects of HSP72 on the MLK3-MKK7-JNK3 module MLK3 phosphorylation is induced during reperfusion with one peak at 30 min and another one at 6 h of reperfusion following 15 min of ischemia, and the latter may account for delayed neuronal death, because JNK signaling activation induced by MLK3 is sufficient to lead to neuronal death through MLK3-MKK7-JNK3 module [29]. In order to investigate effects of HSP72 on the assembly of MLK3-MKK7JNK3 module, we examined changes of the co-precipitation of MLK3MKK7 and MKK7-JNK3 at 6 h of reperfusion following 15 min of ischemia in hippcoampal CA1 regions. We observed that the levels of MLK3 binging to MKK7 and MKK7 binging to JNK3 induced by cerebral I/R were decreased by heat shock, and HSP72-AON could remove these effects (Fig. 4 B). Fig. 4 (A) showed that the lane marked input were all detected followed by immunoblotting with anti-MLK3, anti-

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Fig. 2. Effects of HS, HSP72-AON and HSP72-NON on the expression of HSP72. The expression of HSP72 was significantly increased by HS compared with control, while HSP72-AON could inhibit expression of HSP72 induced by HS (A). The intensity of the bands was expressed as optical density (O.D.) analysis (B). Data were given as mean ± SD gained from six independent animals in each experiment group. ⁎P b 0.05 vs. sham group, #P b 0.05 vs. HS group.

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Fig. 3. Effects of HSP72 on the phosphorylation of Akt1 at 0 min and 3 days in hippocampus by immunoblot analysis after I/R. The phosphorylation of Akt1(ser473) was significantly increased by heat shock compared with I/R control, while HSP72-AON could inhibit phosphorylation of Akt1 induced by heat shock (A). The intensity of the bands was expressed as optical density (O.D.) analysis (B). Data were given as mean ± SD gained from six independent animals in each experiment group. ⁎P b 0.05 vs. sham group, # P b 0.05 vs. I/R group.

MKK7 and anti-JNK3. While immunoprecipitated with nonspecific mouse IgG, no significant band corresponding to MLK3, MKK7 and JNK3 were detected. These data indicated that HSP72 could inhibit the assembly of MLK3-MKK7-JNK3 module.

3.4. Effects of HSP72 on the activation of JNK3, c-Jun and caspase-3 Previous researches indicate that the activation of JNK3 accounts for delayed neuronal death at 3 day of reperfusion following 15 min of ischemia in hippcoampal CA1 regions [33]. We examined the effect of HSP72 on JNK3. As shown in Fig. 5, heat shock reduced the phosphorylation of JNK3, whereas HSP72-AON removed this effect. Moreover, since we have previously examined that c-Jun, a nuclear substrate of JNK3, was activated after reperfusion and reached its activation peak at 6 h of reperfusion [34], it is essential to address the effect of HSP72 on the phosphorylation of c-Jun. It was found that heat shock prevented the increased c-Jun phosphorylation at 6 h of reperfusion. On the contrary, HSP72-AON significantly reversed this effect (Fig. 5). Furthermore, caspase-3 has only one activated peak at 6 h of reperfusion in our previous studies [35]. As shown in Fig. 5, we found that pretreatment with heat shock rescued the I/R induced activation of caspase-3. However, pretreatment with HSP72-AON abscised the effect of heat shock on the caspase-3. It is concluded that

D. Qi et al. / Journal of the Neurological Sciences 317 (2012) 123–129

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Reperfusion for 6 h Fig. 4. Immunoprecipitation analysis of the interaction of MLK3 and MKK7 and the interaction of MKK7 and JNK3. Total proteins were immunoprecipitated with nonspecific mouse IgG (nsIgG), and the precipitates were analyzed by immunoblotting with antiMLK3, anti-MKK7 and anti-JNK3. In the lane marked input, proteins without immunoprecipitation were loaded (A). The interaction between MLK3 and MKK7 and the interaction between MKK7 and JNK3 were all significantly inhibited by heat shock compared with I/R control, while HSP72-AON could remove the inhibition (B). The intensity of the bands was expressed as optical density (O.D.) analysis (C). Data were given as mean ± SD derived from six independent animals in each experiment group. ⁎P b 0.05 vs. sham group, #P b 0.05 vs. I/R group.

HSP72 could decreased the activation of JNK3, c-Jun and caspase-3 induced by cerebral I/R. 4. Discussion As we all know, survival and death of neurons depend on the balance between pro-survival and pro-apoptosis signaling pathways. This balance may be destroyed and lead to apoptosis by some stressful treatments, such as ethanol, UV irradiation, doxorubicin (Adriamycin), amd tumor necrosis factor (TNF). Akt pathway is an important pro-survival signaling pathway [36], while the JNK3 pathway is a momentous proapoptosis signaling pathway. Our current results showed that the

Fig. 5. Effects of HSP72 on the activation of JNK3, c-Jun and cleaved caspase-3. The phosphorylation of JNK3 was significantly inhibited by heat shock compared with I/R control after 3 days reperfusion, while HSP72-AON could remove the inhibition (A). The phosphorylation of c-Jun and the expression of cleaved caspase-3 were attenuated by heat shock after 6 h reperfusion compared with I/R control. HSP72-AON could reverse this effect (A). The intensity of the bands was expressed as optical density (O.D.) analysis (B). Data were expressed as mean ± SD gained from six independent animals in each experiment group. ⁎P b 0.05 vs. sham group, #P b 0.05 vs. I/R group.

decreased Akt1 activation was caused at the end of 15 min of ischemia, subsequently resulting in neuronal damage [37]. On the other hand, the increased Akt1 activation by a variety of stressful pretreatments could participate in endogenous neuroprotection [38]. The mechanisms that a variety of stressful pretreatments leading to the increase of Akt1 activation rescue cells from apoptosis have been extensively studied. But little is known about the involvement of Akt in hyperthermia-induced HSP72 expression in the brain. In order to elucidate the possible mechanism of phosphorylation of Akt mediated by HSP72, we directly injected specific antisense oligonucleotides against HSP72 into the brain ventricles to negatively regulate HSP72 expression in MCAO mice. Here, Our principal findings can be summarized: (1) Hyperthermia-induced HSP72 expression could increase phosphorylation of Akt1(ser473) (Fig. 3) and protect hippocampal neurons from ischemic injury (Fig. 1), (2) combination of MLK3, MKK7 and JNK3 were remarkably downregulated by HSP72 overexpression (Fig. 4), (3) HSP72 overexpression significantly decreased JNK3 phosphorylation, c-Jun phosphorylation and caspase-3 activition (Fig. 5). In a word, preconditioning with heat

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(09KJB310016, 11KJB310013), the National Natural Science Foundation of China (30800446, 31100762 and 30801125), the Key Laboratory of Brain Disease Bioinformation of Jiangsu Province (JSBL1102, JSBL0903), the Special Foundation of President of Xuzhou Medical College (2010KJZ27), and the Qing Lan Project of Jiangsu Province. References

Fig. 6. I/R could suppress Akt1 activation and activates the MAPK signaling pathway and then lead to neuronal apoptosis via MLK3-MKK7-JNK3 module. If Akt1 activation was induced, activated Akt1 could negatively regulate JNK3 and finally block neuronal apoptosis. Pretreatment with moderate heat shock induces overexpression of HSP72 in brain and neuronal survival by inducing Akt1 activation through JNK3 signaling pathway.

shock inhibited the activations of JNK3, c-Jun and caspase-3 during reperfusion after lethal ischemia through HSP72-mediated activation of Akt1. These results might provide some clues to understand the mechanism underlying ischemia tolerance and to find clinical therapies for stroke using the endogenous neuroprotection. HSPs are known to play crucial roles in cytoprotection against environmental stresses like heat or hypoxia. As molecular chaperones, HSPs are important for myofibrillar organization, contractile properties and protection of cerebral I/R [39]. MLK3, an upstream mediator of JNK, is significantly activated after I/R and activated MLK3 phosphorylates MKK7, and then JNK3 is mediated by the MLK3-MKK7-JNK3 signalling module [40]. In this study, we found that the combinations of MLK3 and MKK7, MKK7 and JNK3 were inhibited remarkablely by HSP72. Activation of JNK3 and c-Jun were negatively regulated as well. JNK3 is an important signaling protein in neuron apoptosis, which is activated significantly in cerebral I/R with two peaks at 30 min and 3 days after reperfusion. c-Jun, which is directly downstream of JNK3 and initiates transcription and expression of apoptosis proteins, is also involved in ischemic neuronal death. Additionally, we found that specific antisense oligonucleotides against HSP72 reversed all of the effects of heat shock treatment. Consequently, these studies suggest that there was a close relationship between strengthened Akt1 activation and suppressed JNK3 and c-Jun activation, which was mediated by HSP72. Previous studies suggested that caspase-3 is key in the execution process of apoptosis, and inhibition of its activation can prevent cell from apoptotic death [41]. Our studies proved that HSP72 could decrease the activation of caspase-3, which might take effect at the final step of the apoptotic cascade. For the first time, this work demonstrated that Akt1 activation is involved in the neuroprotection of HSP72 against ischemic brain injury via suppressing JNK3 proapoptosis signaling pathway. At the same time, elevated caspase-3 activation was suppressed by HSP72 to prevent delayed neuronal death in CA1 region. Taking together, we investigated neuroprotective effect of HSP72 and shed light on the underlying signaling mechanism, through which HSP72 expression induced by moderate heat shock could inhibite JNK3 activation through Akt1 pathway by inhibiting the MLK3-MKK7-JNK3 cascade (Fig. 6). It is important that our results provided a new understanding of the function of HSP72 in post-ischemic injury and offer a potential target to acute cerebral ischemia. Acknowledgments This work was supported by the Education Departmental Natural Science Research Funds of Jiangsu Provincial Higher School of China

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