Akt during cerebral ischemia in rat hippocampus

Neuroscience Letters 404 (2006) 98–102

Down-regulation of PTEN by sodium orthovanadate inhibits ASK1 activation via PI3-K/Akt during cerebral ischemia in rat hippocampus Dong-Na Wu, Dong-Sheng Pei, Qing Wang, Guang-Yi Zhang ∗ Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, No. 84 West Huai-hai Road, Xuzhou 221002, Jiangsu, PR China Received 17 January 2006; received in revised form 8 May 2006; accepted 9 May 2006

Abstract In this study, we examined the phosphorylation of ASK1, Akt and PTEN and the effects of sodium orthovanadate on these signal proteins during ischemia. Transient (15 min) brain ischemia was induced by the four-vessel occlusion in Sprague–Dawley rats. The following results were observed: (1) the decreased tyrosine phosphorylation of PTEN and the decreased serine phosphorylation of Akt induced by ischemia were suppressed by sodium orthovanadate, respectively. (2) The phosphorylation of ASK1 at serine 83 was decreased and the phosphorylation of ASK1 at threonine 845 was increased during ischemia. Sodium orthovanadate could alter the phosphorylation status of ASK1 at serine 83 and threonine 845 induced by ischemia. However, LY294002 could reverse the effect of sodium orthovanadate on the phosphorylation of ASK1 at threonine 845, namely, sodium orthovanadate inhibited ASK1 through the PI3-K/Akt-dependent pathway. Taken together, we concluded that sodium orthovanadate could increase the tyrosine posphorylation of PTEN and further inhibit the activation of ASK1 via activating Akt during cerebral ischemia. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: PTEN; Akt; ASK1; Brain ischemia; Phosphorylation; Sodium orthovanadate

Global ischemia occurs in the case of cardiac arrest in the clinical setting, and transient global ischemia results in several pathophysiological changes that may be implicated in the regulation of multiple survival and death-signaling pathway. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor gene localized to chromosome 10q23. The PTEN protein is both a protein and a lipid phosphatase [5,13]. The lipid phosphatase activity of PTEN can dephosphorylate the D3 position of PtdIns 3,4-P2 and PtdIns 3,4,5-P3, the lipid products of the PI3-K lipid kinase activity, thus, PTEN antagonizes PI3-K/Akt signaling pathway by counteracting the activity of PI3-K [12,13]. Recent studies demonstrate that the protein phosphatase activity of PTEN can regulate cell migration, spreading, and growth [16]. However, little is known about the role of PTEN/PI3-K pathway in the central nervous system (CNS).

∗

Corresponding author. Tel.: +86 516 5748486; fax: +86 516 5748486. E-mail address: [email protected] (G.-Y. Zhang).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.05.018

Akt, also known as protein kinase B (PKB), is a critical mediator involved in survival signals as a downstream kinase of phosphoinositide 3-kinase (PI3-K) in growth factor-mediated signaling cascades. Phosphorylation of residues Thr-308 and Ser-473 is required for Akt activity [8,18,19]. There is convincing evidence that Ser-473 is phosphorylated by S473K and Thr308 is phosphorylated by 3-phosphoinositol-dependent kinase 1 (PDK1) [1,6]. Active Akt phosphorylates BAD, caspase-9, glycogen synthase kinase 3 (GSK-3), ASK1, fork-head transcription factors, and thereby induces antiapoptotic effects [8,18]. ASK1 (apoptosis signal-regulating kinase 1) is a mitogenactivated protein kinase kinase kinase that phosphorylates and activates mitogen-activated protein kinase kinase 4 (MKK4) or MKK7 and MKK3 or MKK6, which in turn activates the stress-activated protein kinase c-Jun N-terminal kinase (JNK) and p38 pathways [3,8]. Phosphorylation of ASK1 at Ser-83 (a specific Akt phosphorylation site) is correlated with the decreased activity of ASK1. The threonine 845 of ASK1 is an autophosphorylation site and its phosphoryla-

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tion is required for the activation of ASK1 [17]. A variety of stress-related stimuli can activate ASK1, including serum or trophic factor withdrawal, TNF-␣, reactive oxygen species (ROS), genotoxic stress, ischemia insult, and possibly Fasl [2,4]. Orthovanadate is a phosphate analog and generally thought to bind as a transition state analog to the phosphoryl transfer enzymes and inhibit ATPases such as phosphatases, including acid or alkaline phosphatases, and phosphoprotein tyrosine phosphatases [9]. Thus, orthovanadate can affect intracellular tyrosine phosphorylation levels via inhibition of non-selective protein tyrosine phosphatases. In the present study, we investigated the phosphorylation of PTEN, Akt and ASK1 during ischemia. Sodium orthovanadate was used to explore the mechanism underlying the regulation between PI3-K/PTEN/Akt pathway and the activation of ASK1 during brain ischemia. Adult male Sprague–Dawley (SD) rats (Shanghai Experimental Animal Center, Chinese Academy of Science) weighing 250–300 g were subjected to 5, 15, and 30 min of brain ischemia by four-vessel occlusion method as described before [15]. Sham control received the same surgical procedure, except for occlusion of carotid artery. When necessary, sodium orthovanadate (15 mg/kg, dissolved in saline, sigma) or the same dose of vehicle (saline) was administrated to the rats by abdominal injection 20 min prior to cerebral ischemia. Rats were decapitated immediately after ischemia, and the hippocampus tissues were removed and frozen in liquid nitrogen. The hippocampus tissues were homogenized in ice-cold homogenization buffer containing 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS) (Sigma; pH 7.4), 100 mM KCl, 320 mM sucrose, 50 mM NaF, 0.5 mM MgCl2 , 0.2 mM DTT, 1 mM EDTA, 1 mM EGTA, 1 mM Na3 VO4 (Sigma), 20 mM sodium pyrophosphate, 20 mM ␤-glycerophosphate, 1 mM p-nitrophenyl phosphate (PNPP), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 ␮g/ml each of leupeptin, aprotinin, pepstatin A. The homogenizations were centrifuged at 800 × g for 15 min at 4 ◦ C. The supernatants were collected and protein concentration was determined by the method of Lowry. The immunoprecipitation and immunoblot were performed with specific antibodies as described by Liu et al. [11]. After immunoblot, the bands on the membrane were scanned and analyzed with an image analyzer (LabWorks Software, UVP Inc., Upland, CA, USA). Rabbit polyclonal anti-p-Akt (Ser-473), rabbit polyclonal anti-Akt, rabbit polyclonal anti-p-ASK1 (Ser-83), rabbit polyclonal anti-p-ASK1 (Thr-845), rabbit polyclonal anti-ASK1, and rabbit polyclonal anti-PTEN were purchased from cell signaling biotechnology. Phosphotyrosine (clone PT-66) was from sigma. Values were expressed as mean ± S.D. from four independent rats. Statistical analysis of the results was performed by one-way analysis of variance followed by the Duncan’s new multiple range method. P < 0.05 was considered significant. As an upstream regulator of Akt, the phosphatase PTEN may play a role in the regulation of survival signaling during the process of neuronal injury induced by cerebral ischemia. In order to

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Fig. 1. PTEN is phosphorylated at the tyrosine residue in hippocampus. Homogenates (400 ug of total protein) from sham-operated control and ischemia animal at 15 min were immunoprecipitated with anti-PTEN, anti-pY, or nonspecific IgG, and the precipitates were analyzed by immunoblotting with anti-PTEN or anti-pY. In the lane marked input, 100 ug of protein without immunoprecipitation were loaded. Bands corresponding to PTEN were scanned and the intensities were represented as folds vs. sham control. a P < 0.05 vs. sham.

elucidate the regulation of PI3-K/Akt pathway by phosphatase PTEN during cerebral ischemia, we firstly examined the phosphorylation of PTEN and Akt at various time points of ischemia. Immunoprecipitation and immunoblot were used to examine tyrosine phosphorylation of PTEN during ischemia in rat hippocampus. As shown in Fig. 1, either immunoprecipitation with anti-PTEN antibody followed by blot with anti-phosphotyrosine or immunoprecipitation with anti-phosphotyrosine antibody followed by blot with anti-PTEN, a band of 54 kDa was detected. And a band of the same molecular weight in the lane marked input was also detected. The results showed that PTEN was phosphorylated at tyrosine residue and the tyrosine phosphorylation of PTEN from sham-operated controls was higher than that from ischemia group. Our results also showed that the tyrosine phosphorylation of PTEN declined in a time-dependent manner during ischemia, whereas the phosphorylation of PTEN (Ser380 /Thr382/383 ) and the protein levels of PTEN had no remarkable change at each time point (Fig. 2A and B). Previous study indicated that the tyrosine phosphorylation of PTEN could decrease the ability of PTEN to hydrolyze PtdIns and reduce PTEN stability [12], thus, we speculated that the ischemia insult might affect the ability of PTEN to hydrolyze PtdIns. Consis-

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Fig. 2. Effects of sodium orthovanadate on the tyrosine phosphorylation of PTEN: (A) time course of the expression and phosphorylation on tyrosine of PTEN during ischemia in rat hippocampus; (B) quantitative representation of the expression and phosphorylation of PTEN during ischemia; (C) effects of sodium orthovanadate on the tyrosine phosphorylation of PTEN at 15 min of ischemia; (D) quantitative representation of the effects of sodium orthovanadate on the tyrosine phosphorylation of PTEN. Data were expressed as mean ± S.D. of four independent animals. a P < 0.05 vs. sham. b P < 0.05 vs. I30 min +saline. SV15: sodium orthovanadate of 15 mg/kg was administrated to the rats 20 min before ischemia.

Fig. 3. Effects of sodium orthovanadate and LY294002 on the phosphorylation of Akt: (A) time course of the expression and phosphorylation of Akt during ischemia in rat hippocampus; (B) quantitative representation of the expression and phosphorylation of Akt during ischemia; (C) effects of sodium orthovanadate and LY294002 on the expression and phosphorylation of Akt at 15 min of ischemia in hippocampus; (D) quantitative representation of the effects of sodium orthovanadate and LY294002 on the expression and phosphorylation of Akt. Data represent the mean ± S.D. of four independent animals. a P < 0.05 vs. sham, b P < 0.05 vs. I30 min +saline, c P < 0.05 vs. LY294002 + SV15. SV15: sodium orthovanadate of 15 mg/kg was administrated to the rats 20 min before ischemia.

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Fig. 4. Effects of sodium orthovanadate and LY294002 on the phosphorylation of ASK1: (A) time course of the expression and phosphorylation of ASK1 during ischemia in rat hippocampus; (B) quantitative representation of the expression and phosphorylation of ASK1 during ischemia; (C) effects of sodium orthovanadate and LY294002 on ASK1 activity at 15 min of ischemia in hippocampus; (D) quantitative representation of the effects of sodium orthovanadate and LY294002 on the expression and phosphorylation of ASK1. Data were expressed as mean ± S.D. of four independent animals. a P < 0.05 vs. sham, b P < 0.05 vs. I30 min +saline, c P < 0.05 vs. LY294002 + SV15. SV15: sodium orthovanadate of 15 mg/kg was administrated to the rats 20 min before ischemia.

tent with our presumption, the decreased activity of Akt was observed in response to cerebral ischemia (Fig. 3A and B). After pretreatment with sodium orthovanadate, the tyrosine phosphorylation of PTEN was increased at 15 min of ischemia (Fig. 2C and D). Meanwhile, the activity of Akt was also increased significantly (Fig. 3C and D). Previous studies demonstrated that the down-regulation of PTEN could play a neuroprotective role [7,10,14], our study also provided evidence that the alteration of PTEN function might be implicated in the regulation of Akt survival pathway during ischemia. The effects of sodium orthovanadate on PI3-K/Akt signaling pathway appeared to be due, at least in part, to an alteration of PTEN activity mediated by phosphorylation on tyrosine residue. Akt is a crucial mediator of neuronal survival. Although the mechanisms by which Akt signaling inhibits apoptosis are still emerging, findings indicate that Akt phosphorylates and disables components of the apoptotic machinery, including ASK1 [8]. In our present study, we examined the activity of ASK1 during ischemia. As shown in Fig. 4A and B, we found that the phosphorylation of ASK1 at Ser-83 was diminished and at the same time, the phosphorylation of ASK1 at Thr-845 dramatically increased. It has been shown that Akt can phosphorylate ASK1 at Ser-83 to maintain ASK1 in an inactive form. However, Thr-845 is an autophosphorylation site that is critical for ASK1 activation, its phosphorylation leads to an active form of ASK1 [8,17]. Our present study indicated that ASK1 was activated in response to ischemia. It had been reported that H2 O2 could induce the

decreased phosphorylation of Akt and ASK1 (Ser-83), while the phosphorylation of ASK1 at Thr-845 was increased. Moreover, the same phenomenon was observed in Akt− /− MEF [19]. Although the mechanism underlying the activation of ASK1 in response to apoptotic stimuli has not been fully elucidated, from above presented evidence, we could deduce that phosphorylation of ASK1 at Ser-83 antagonize the phosphorylation at Thr-845. To further examine whether Akt is involved in the regulation of ASK1 activity during ischemia, we observed the effects of sodium orthovanadate and LY294002 (a specific inhibitor of PI3-K) on the activation of ASK1 (shown in Fig. 4C and D). Our results showed that sodium orthovanadate decreased the activation of ASK1, whereas pretreatment with sodium orthovanadate and LY294002 simultaneously could block the alteration of the phosphorylation of ASK1 induced by sodium orthovanadate, which suggested that sodium orthovanadate inhibited ASK1 through the PI3-K/Akt-dependent pathway. The above results indicate that the increase of PTEN phosphotyrosine by sodium orthovanadate inhibits the activation of ASK1 via activating Akt during cerebral ischemia. In summary, sodium orthovanadate can activate the PTEN/PI3-K/Akt signaling pathway and further inhibit ASK1 activity, which enable sodium orthovanadate function as an inhibitor of apoptosis. In addition, our study provided evidence that down-regulation of PTEN function resulted in the increased activity of Akt and the decreased activity of its downstream target ASK1 during ischemia, which indicated that down-

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regulation of PTEN might be an effective way in the treatment of stroke. Acknowledgment This work was supported by a grant from the Key Project of the National Natural Science Foundation of China (grant no. 30330190). References [1] D.R. Alessi, S.R. James, C.P. Downes, A.B. Holmes, P.R. Gaffney, C.B. Reese, P. Cohen, Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha, Curr. Biol. (1997) 261–269. [2] H.Y. Chang, H. Nishitoh, X. Yang, H. Ichijo, D. Baltimore, Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx, Science 281 (1998) 1860–1863. [3] S.J. Charette, H. Lambert, J. Landry, A kinase-independent function of ASK1 in caspase-independent cell death, J. Biomed. Chem. 276 (2001) 36071–36074. [4] Z. Chen, H. Seimiya, M. Naito, T. Mashima, A. Kizaki, S. Dan, M. Imaizumi, H. Ichijo, K. Miyazono, T. Tsuruo, ASK1 mediates apoptotic cell death induced by genotoxic stress, Oncogene 18 (1999) 173– 180. [5] S.H. Cho, C.H. Lee, Y. Ahn, H. Kim, H. Kim, C.Y. Ahn, K.S. Yang, S.R. Lee, Redox regulation of PTEN and protein tyrosine phosphatases in H(2) O(2) mediated cell signaling, FEBS Lett. 560 (2004) 7–13. [6] J. Feng, J. Park, P. Cron, D. Hess, B.A. Hemmings, Identification of a PKB/Akt hydrophobic motif ser-473 kinase as DNA-dependent protein kinase, J. Biol. Chem. 279 (2004) 41189–41196. [7] D.S. Gary, M.P. Mattson, PTEN regulates Akt kinase activity in hippocampal neurons and increases their sensitivity to glutamate and apoptosis, Neuromolecular Med. 2 (2002) 261–269. [8] A.H. Kim, G. Khursigara, X. Sun, T.F. Franke, M.V. Chao, Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1, Mol. Cell Biol. 21 (2001) 893–901.

[9] F. Kohji, K. Takayuki, Akt is a molecular target for signal transduction therapy in brain ischemic insult, J. Pharmacol. Sci. 92 (2003) 317–327. [10] J.H. Lee, K.Y. Kim, Y.K. Lee, S.Y. Park, C.D. Kim, W.S. Lee, Cilostazol prevents focal cerebral ischemic injury by enhancing casein kinase 2 phosphorylation and suppression of phosphatase and tensin homolog deleted from chromosome 10 phosphorylation in rats, J. Pharmacol. Exp. Ther. 308 (2004) 896–903. [11] Y. Liu, G.Y. Zhang, C. Gao, X.Y. Hou, NMDA receptor activation results in tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and interaction of Pyk2 and Src with NR2A after transient cerebral ischemia and reperfusion, Brain Res. 909 (2001) 51–58. [12] Y. Lu, Q. Yu, J.H. Liu, J. Zhang, H. Wang, D. Koul, J.S. Mcmurray, X. Fang, W.K. Alferyung, K.A. Siminovitch, Src family protein–tyrosine kinases alter the function of PTEN to regulate phosphatidylionsitol 3kinase/AKT cascades, J. Biom. Chem. 278 (2003) 40057–40066. [13] M.P. Myers, J.P. Stolarov, C. Eng, J. Li, S.I. Wang, M.H. Wigler, R. Parsons, N.K. Tonks, P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatases, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 9052–9057. [14] K. Ning, L. Pei, M. Liao, B. Liu, Y. Zhang, W. Jiang, J.G. Mielke, L. Li, Y. Chen, Y.H. El-hayek, M.G. Fenhlings, X. Zhang, F. Liu, J. Eubanks, Q. Wan, Dual Neuroprotective signaling mediated by downregulating two distant phosphatase activities of PTEN, J. Neurosci. 24 (2004) 4052–4060. [15] W.A. Pulsinelli, J.B. Briverley, A new moldel of bilateral hemispheric ischemia in the unanesthetized rat, Stroke 10 (1979) 262–272. [16] M. Tamura, J. Gu, T. Takino, K.M. Yamada, Tumor suppressor PTEN inhibitor of cell invision, migration, and growth: differential involvement of focal adhesion kinase and p130Cas, Cancer Res. 59 (1999) 442–449. [17] K. Tobiume, M. Saitoh, H. Ichijo, Activation of apoptosis signalregulating kinase 1 by the stress-induced activating phophorylation of pre-formed oligomer, J. Cell Physiol. 191 (2002) 95–104. [18] S. Yano, M. Morioka, K. Fukunaga, T. Kawano, T. Hara, Y. Kai, J. Hamada, E. Miyamoto, Y. Ushio, Activation of Akt/Protein Kinase B contributes to induction of ischemic tolerance in the CA1 subfield of gerbil hippocampus, J. Cereb. Blood Flow. Metab. 21 (2001) 351–360. [19] R. Zhang, D. Luo, R. Miao, L. Bai, Q. Ge, W.C. Sessa, W. Min, Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosis, Oncogene 24 (2005) 3954–3963.