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Region-specific phosphorylation of ATF-2, Elk-1 and c-Jun in rat hippocampus and cerebellum after electroconvulsive shock
Neuroscience Letters 329 (2002) 9–12 www.elsevier.com/locate/neulet
Region-specific phosphorylation of ATF-2, Elk-1 and c-Jun in rat hippocampus and cerebellum after electroconvulsive shock Yong Min Ahn a, Ung Gu Kang b, Seung Wook Oh c, Yong-Sung Juhnn c, Yeon-Ho Joo d, Joo-Bae Park e, Yong Sik Kim b,* a
Department of Neuropsychiatry, Eulji University School of Medicine, Seoul 139-711, South Korea Department of Psychiatry, Seoul National University College of Medicine, Clinical Research Institute, Seoul National University Hospital, and Institute for Neuroscience, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea c Department of Biochemistry, Seoul National University College of Medicine, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea d Department of Psychiatry, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, South Korea e Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea b
Received 24 April 2002; accepted 13 May 2002
Abstract There have been reports of regional differences in the activation of mitogen activated protein kinases (MAPKs) and in the induction of immediate early genes after electroconvulsive shock (ECS) in the rat brain. This study was performed to determine whether ECS induce the region-specific phosphorylation of MAPK-downstream transcription factors, ATF-2, Elk-1, c-Jun, in rat hippocampus and cerebellum. Following ECS, the phosphorylation of ATF-2 was highly increased in the hippocampus but slightly in the cerebellum. The phosphorylation of Elk-1 was increased in the cerebellum but not in the hippocampus. In contrast, the phosphorylation of c-Jun was increased only in the hippocampus. These results indicate that ECS can induce the region-specific phosphorylation of MAPK-downstream transcription factors in rat hippocampus and cerebellum. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Electroconvulsive shock; Hippocampus; Cerebellum; ATF-2, Elk-1, c-Jun
Electroconvulsive shock (ECS) induces the expression of the immediate early genes (IEGs) such as c-fos and tetradecanoyl phorbol acetate-inducible sequences (TIS) in the rat brain [4,9]. Previously, we have reported that the induction patterns of IEGs after ECS vary depending on the brain regions. For example, TIS genes were induced more rapidly in the cerebellum than in the hippocampus [9]. Our previous experiments also show that the activation of mitogen activated protein kinases (MAPKs; ERK1/2, JNKs and p38) after ECS is different between the hippocampus and the cerebellum [11,14]. The phosphorylation of ERK1/2, p38 and JNKs was clearly increased after ECS in the hippocampus. However, in the cerebellum, only p38 showed an increased phosphorylation that had intensity comparable to that in the hippocampus. This study was conducted to determine whether ECS could induce the region-specific * Corresponding author. Tel.: 182-2-760-2204; fax: 182-2-7447241. E-mail address: [email protected] (Y.S. Kim).
phosphorylation of MAPK-downstream transcription factors, ATF-2, Elk-1, c-Jun, three important regulatory proteins for the induction of IEGs, in rat hippocampus and cerebellum. The activation of ATF-2 requires the phosphorylation at Thr69 and Thr71, which can be mediated by JNKs and p38 [5,15]. The phosphorylation at Ser383 and Ser389 of Elk-1 is critical for its transcriptional activity and can be accomplished by ERKs, JNKs and p38 [17]. Transcription factor cJun is regarded as a kind of IEGs and its expression is regulated by various stresses in neuronal and non-neuronal cell [13]. The transcriptional activity of c-Jun is regulated by the phosphorylation at Ser63 and Ser73, which is catalyzed by JNKs [13]. Recent data suggest that c-Jun is phosphorylated by other kinases as well, e.g. ERKs [7]. These associations between MAPKs and transcription factors have mostly emerged from cell culture models and it is difficult to extrapolate them directly to the brain in vivo [1,14]. However, very few studies have been conducted concerning the activation of transcription factors following
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ECS in vivo. Bhat et al. [1] reported that the phosphorylation of Elk-1 was increased in the cerebral cortex but not in the hippocampus after ECS. Brecht et al. [2] reported an increase in the nuclear phosphorylated c-Jun after ECS in many brain regions. Nevertheless, these studies [1,2] were about the activation of a single transcription factor and thus did not pursue the regional differences in the activation of signal transduction system. Male Sprague–Dawley rats (150–200 g) were used in this study. Throughout the entire procedure animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Electroconvulsive shock was administered to the animals via ear-clip electrodes (130 V, 0.5 s, Medicraft B-24). Control animals were identically handled except they did not receive any electric current (sham treatment). The rats were decapitated at 0, 1, 2, 5, 10, 30, 60, and 120 min after ECS. The hippocampus and the cerebellum were homogenized in ten volumes (v/w) of pre-chilled buffer containing 25 mM HEPES (pH 7.9), 200 mM NaCl, 1.5 mM MgCl2, 0.2% NP-40, 1 mM DTT, 0.5 mM EDTA, 1 mM PMSF, 20 mM beta-glycerophosphate, 2 mM NaF, 0.1 mM Na3VO4, and 2 mg/ml leupeptin. The Homogenates were centrifuged and the supernatants were boiled with Laemmli’s sample buffer. Immunoblotting was performed using anti-phospho-ATF-2 (Thr71), anti-phospho-Elk-1 (Ser383) and anti-phospho-c-Jun (Ser73) antibodies (New England Biolab.). Signals were detected using ECL system (Pierce). The membranes were then reprobed with anti-ATF-2, anti-Elk-1 and anti-c-Jun antibodies (New England Biolab.) to determine the amounts of the proteins. After confirming the temporal pattern of the phosphorylation, six independent sets of samples were prepared at 2 and 30 min after ECS and statistical analysis was performed. Immunoblot data were processed by the densitometer and the paired t-test was performed on the quantified data. The criterion for significance was P , 0:05. ECS caused a substantial phosphorylation of ATF-2 in both the hippocampus and the cerebellum without altering the protein amount of ATF-2. The phosphorylation started to increase at 1 min and reached peak at about 5 min after ECS. Then it decreased slowly, but was still above the basal level at 120 min after ECS in the hippocampus and the cerebellum (Fig. 1A). At 2 min after ECS, there was a statistically significant increase in the phosphorylation of ATF-2 compared to the sham condition in the hippocampus (P ¼ 0:040) and the cerebellum (P ¼ 0:039) (Fig. 1B). The level of increased phosphorylation in the hippocampus was greater than that in the cerebellum (210.5 ^ 63.6 versus 122.0 ^ 19.4%). At 30 min after ECS, this statistical difference from the sham control was not observed (Fig. 1B). In the cerebellum, ECS increased the phosphorylation of Elk-1 without changing the total protein amount (Fig. 1A). Two minutes after ECS, at which time the phosphorylation of Elk-1 reached peak level, the intensity of phospho-Elk-1 increased by 205% compared to the basal level (P ¼ 004; Fig. 1B). The basal level of Elk-1 phosphorylation was
recovered at 30 min after ECS (Figs. 1A,B). However, neither the protein amount nor the phosphorylation of Elk1 increased in the hippocampus after ECS (Figs. 1A,B). The phosphorylation of c-Jun in the hippocampus began to increase at 1 min, reached peak level at 30–60 min, and did not return to its basal level even at 120 min after ECS (Fig. 1A). In contrast to ATF-2 and Elk-1, the protein amount of c-Jun in the hippocampus was also increased by ECS. The expression of c-Jun protein started to increase at 30 min and continued to increase until 120 min (Fig. 1A). ECS resulted in significant increase of both phosphorylation (174.9 ^ 57.3%, P ¼ 0:043) and protein amount (222.7 ^ 41.5%, P ¼ 0:010) when analyzed at 30 min after ECS (Fig. 1B). Therefore, increased protein amount itself might contribute to the increase in phosphorylation of c-Jun at 30 min after ECS. Whereas, at 2 min after ECS, the phosphorylation of c-Jun (143.2 ^ 36.4%) tended to increase, but did not reach statistical significance (P ¼ 0:056). At these earlier time points, the increased level of phosphorylated c-Jun can not have resulted from the increase in the protein amount, because there was not enough time for protein synthesis (Figs. 1A,B). In contrast to the hippocampus, there was no significant increase in the amount and phosphorylation of c-Jun in the cerebellum (Figs. 1A,B). In this study, ECS induced region-specific phosphorylation of MAPK-downstream transcription factors in rat hippocampus and cerebellum. This finding is supported by the observation that ECS resulted in the phosphorylation of ATF-2 and c-Jun in the hippocampus compared to the phosphorylation of ATF-2 and Elk-1 in the cerebellum. The mechanism leading to the selective phosphorylation of transcription factor is still uncertain, but it is possible that the selective phosphorylation of transcription factors may result from the region-specific activation of MAPKs. The activation of JNKs and p38 leads to the phosphorylation of ATF-2 in cell lines [5,15], and our previous study showed that ECS activated JNKs and p38 in rat hippocampus but activated only p38 in the cerebellum [14]. Therefore, the phosphorylation of ATF-2 in the hippocampus after ECS seems to parallel the activation of JNK and p38. However, in the cerebellum, a slightly increased phosphorylation of ATF-2 after ECS may result from the activation of p38 alone. Although all members of MAPKs were activated in the hippocampus [11,14] and all members of MAPKs phosphorylate Elk-1 in cell culture system [17], the increase in the phosphorylation of Elk-1 was not detected after ECS in the hippocampus. The lack of the phosphorylated Elk-1 in the hippocampus after ECS was also reported by Bhat et al. [1]. They explained that the lack of Elk-1 phosphorylation in the hippocampus might result from the limited localization of the activated ERKs to the cytoplasm, and not the nucleus. But MAPKs other than ERKs (i.e. JNK and p38) known to phosphorylate Elk-1, were also activated in the hippocampus after ECS [14,17] and their downstream transcription factors such as ATF-2 and c-Jun were also phosphorylated
Y.M. Ahn et al. / Neuroscience Letters 329 (2002) 9–12
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Fig. 1. The phosphorylation of AFT-2, Elk-1, and c-Jun in rat hippocampus and cerebellum after ECS. (A) The representative blots showing the time course of the phosphorylation of ATF-2, Elk-1 and c-Jun in the hippocampus and cerebellum after ECS. S indicates sham-treated rats that did not receive the electric current. Time (min) indicates the minutes at which the rats were sacrificed after single ECS. (B) The changes in the amount of phosphorylation of ATF-2, Elk-1, c-Jun, and the amount of total c-Jun protein in the hippocampus and cerebellum at 2 and 30 min after ECS. At least six independent experiments were carried out, and the average Relative O.D. (optical densities) was expressed as a percentage of the sham-treated control at ECS 2 or 30 min. The asterisks (*) indicate statistically significant difference compare to sham (P , 0:05, paired t-test).
in the hippocampus in the present study. Therefore the lack of Elk-1 phosphorylation in the hippocampus is not fully explained by the limited localization of ERKs to the cytoplasm. In the cerebellum, ECS increased the phosphorylation of p38 but not of JNK [14], and the increase in the phosphorylation of ERKs in the cerebellum was far less than that in the hippocampus [11]. So, it is plausible that the phosphorylation of Elk-1 in the cerebellum is mainly due to the activation of p38. The differential activation of c-Jun has been suggested by our previous study [14], which showed that the activity of JNKs, major kinases for c-Jun, was dramatically enhanced in a few minutes after ECS in the hippocampus but not in the cerebellum [14]. There have been recent reports of discrepancies between the phosphorylation of c-Jun and the activity of JNKs [7]. But in our observation, the lack of phosphorylation of c-Jun in the cerebellum can be attributed to the lack of JNK activation in this region. Naturally, we should not presume that the phosphorylation of ATF-2, Elk-1 and c-Jun after ECS is
solely determined by MAPKs, for ECS activates multiple signaling pathways that converge in complex manner to regulate the transcription factors. For instance, we have through previous experiments seen the activation of Rafs in the rat brain after ECS [12], and Elk-1 is known to be phosphorylated by a Raf-dependent but MAPK-independent pathway [3]. Just as the phosphorylation of ATF-2, Elk-1 and c-Jun may not be solely determined by MAPKs, previously reported tissue-specific differences in the induction of IEGs after ECS in the rat brain can not be entirely explained by the results in the present study, since the phosphorylation of transcription factors does not always lead to increase in its transcriptional activity [7]. However, the phosphorylations of transcription factors do suggest a spatiotemporal association with the induction of IEGs. And the regional difference in the phosphorylation of transcription factors may be better grasped by observing interactions among many regulatory elements in the promoter region. ECS
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was found to increase the phosphorylation of CREB, another member of ATF/CREB family transcription factors, in the hippocampus but not in the cerebellum after ECS [8], indicating that ECS may induce the IEGs expression in the hippocampus through activation of the CRE motif. c-Jun can also bind to the CRE motifs when forming a heterodimer with ATF-2 [6]. The simultaneous phosphorylation of the c-Jun and ATF-2 was observed in the hippocampus, but not in the cerebellum, suggesting that region-specific phosphorylation of c-Jun and ATF-2 following ECS in the hippocampus may induce, via CRE, region-specific induction of IEGs. Nurr 1, a growth factor-inducible member of the steroid/thyroid hormone receptor gene superfamily, is another gene that contains CRE but not SRE in the promoter region [16]. There was a report of rapid induction of Nurr 1 by single ECS in rat hippocampus but not in other brain regions including the cerebellum [16]. On the other hand, the signal pathways that activate Elk-1 are a major input pathway for SRE-driven IEGs induction [10], and may play important role in induction of IEGs following ECS in the cerebellum but not in the hippocampus. The promoter region of TIS 8 contains five SREs and the SREs are the dominant regulators of TIS 8 expression [6]. In previous study [9], ECS was found to induce many TIS genes both in the cerebellum and the hippocampus, but the induction level of only TIS 8 messenger RNA (mRNA) among TIS gene was much greater in the cerebellum. The expression of TIS 8 is known to be regulated by other transcription factors as well as Elk-1, which might explain the induction of the gene in the hippocampus in the absence of Elk-1 phosphorylation. But the region-specific phosphorylation of Elk-1 might explain the stronger induction of TIS 8 gene in the cerebellum following ECS. In summary, we have shown that ECS can induce the region-specific phosphorylation of MAPKs-downstream transcription factors in rat hippocampus and cerebellum. The results from this study, in conjunction with the previous reports of region specific activation of MAPKs and induction of IEGs after ECS, suggest that there may be regionspecific difference in the activation of intracellular signal transduction pathway after ECS in the rat brain This study was supported by Seoul National University Hospital Research Fund (03-99-70) and in part by 2002 BK21 project for Medicine, Dentistry and Pharmacy.
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[16] [1] Bhat, R.V., Engber, T.M., Finn, J.P., Koury, E.J., Contreras, P.C., Miller, M.S., Dionne, C.A. and Walton, K.M., Region specific targets of p42/44 MAPK signaling in rat brain, J. Neurochem., 70 (1998) 558–571. [2] Brecht, S., Simler, S., Vergnes, M., Mielke, K., Marescaux, C. and Herdegen, T., Repetitive electroconvulsive seizures induce activity of c-Jun N-terminal kinase and compartment-specific desensitization of c-Jun phosphorylation in the rat brain, Brain Res. Mol. Brain Res., 68 (1999) 101–108. [3] Chung, K.C., Gomes, I., Wang, D., Lau, L.F. and Rosner,
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M.R., Raf and fibroblast growth factor phosphorylate Elk1 and activate the serum response element of the immediate early gene pip92 by mitogen-activated protein kinase-independent as well as -dependent signaling pathways, Mol. Cell. Biol., 18 (1998) 2272–2281. Cole, A.J., Abu-Shakra, S., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid rise in transcription factor mRNAs in rat brain after electroshock-induced seizures, J. Neurochem., 55 (1990) 1920–1927. Hazzalin, C.A., Cano, E., Cuenda, A., Barratt, M.J., Cohen, P. and Mahadevan, L.C., P38/RK is essential for stress-induced nuclear responses: JNK/SAPKs and c-Jun/ATF-2 phosphorylation are insufficient, Curr. Biol., 6 (1996) 1028–1031. Herdegen, T. and Leah, J.D., Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins, Brain Res. Rev., 28 (1998) 370–490. Herdegen, T. and Waetzig, V., AP-1 proteins in the adult brain: facts and fiction about effectors of neuroprotection and neurodegeneration, Oncogene, 20 (2001) 2424–2437. Jeon, S.H., Seong, Y.S., Juhnn, Y.S., Kang, U.G., Ha, K.S., Kim, Y.S. and Park, J.-B., Electroconvulsive shock increases the phosphorylation of cyclic AMP response element binding protein at Ser-133 in rat hippocampus but not in cerebellum, Neuropharmacology, 36 (1997) 411–414. Jung, H.Y., Kang, U.G., Ahn, Y.M., Joo, Y.H., Park, J.-B. and Kim, Y.S., Induction of tetradecanoyl phorbol acetate-inducible sequence (TIS) genes by electroconvulsive shock in rat brain, Biol. Psychiatry, 40 (1996) 503–507. Kaminska, B., Kaczmarek, L., Zangenehpour, S. and Chaudhuri, A., Rapid phosphorylation of Elk-1 transcription factor and activation of MAP kinase signal transduction pathways in response to visual stimulation, Mol. Cell Neurosci., 13 (1999) 405–414. Kang, U.G., Hong, K.S., Jung, H.Y., Kim, Y.S., Soeng, Y.S., Yang, Y.C. and Park, J.-B., Activation and tyrosine phosphorylation of 44-kDa mitogen-activated protein kinase (MAPK) induced by electroconvulsive shock in rat hippocampus, J. Neurochem., 63 (1994) 1979–1982. Kang, U.G., Jeon, S.H., Lee, J.E., Joo, Y.H., Yi, J.S., Park, J.B., Juhnn, Y.S. and Kim, Y.S., The activation of B-Raf and Raf-1 after electroconvulsive shock in the rat hippocampus, Neuropharmacology, 39 (2000) 703–706. Minden, A. and Karin, M., Regulation and function of the JNK subgroup of MAP kinases, Biochim. Biophys. Acta, 1333 (1997) F85–F104. Oh, S.W., Ahn, Y.M., Kang, U.G., Kim, Y.S. and Park, J.-B., Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock, Neurosci. Lett., 271 (1999) 101–104. van Dam, H., Wilhelm, D., Herr, I., Steffen, A., Herrlich, P. and Angel, P., ATF-2 is preferentially activated by stress-activated protein kinase to mediate c-jun induction in response to genotoxic agents, EMBO J., 14 (1995) 1798–1811. Xing, G., Zhang, L., Zhang, L., Heynen, T., Li, X.-L., Smith, M.A., Weiss, S.R.B., Feldman, A.N., Detera-Wadleigh, S., Chuang, D.-M. and Post, R.M., Rat nurr1 is prominently expressed in perihinal cortex, and differential induced in the hippocampal dentate gyrus by electroconvulsive versus kindled seizures, Brain Res. Mol. Brain Res., 47 (1997) 251– 261. Yang, S.-H., Whitmarsh, A.J., Daivs, R.J. and Sharrocks, A.D., Differential targeting of MAP kinase to the ETSdomain transcription factor Elk-1, EMBO J., 17 (1998) 1740–1749.
Region-specific phosphorylation of ATF-2, Elk-1 and c-Jun in rat hippocampus and cerebellum after electroconvulsive shock Yong Min Ahn a, Ung Gu Kang b, Seung Wook Oh c, Yong-Sung Juhnn c, Yeon-Ho Joo d, Joo-Bae Park e, Yong Sik Kim b,* a
Department of Neuropsychiatry, Eulji University School of Medicine, Seoul 139-711, South Korea Department of Psychiatry, Seoul National University College of Medicine, Clinical Research Institute, Seoul National University Hospital, and Institute for Neuroscience, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea c Department of Biochemistry, Seoul National University College of Medicine, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea d Department of Psychiatry, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, South Korea e Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea b
Received 24 April 2002; accepted 13 May 2002
Abstract There have been reports of regional differences in the activation of mitogen activated protein kinases (MAPKs) and in the induction of immediate early genes after electroconvulsive shock (ECS) in the rat brain. This study was performed to determine whether ECS induce the region-specific phosphorylation of MAPK-downstream transcription factors, ATF-2, Elk-1, c-Jun, in rat hippocampus and cerebellum. Following ECS, the phosphorylation of ATF-2 was highly increased in the hippocampus but slightly in the cerebellum. The phosphorylation of Elk-1 was increased in the cerebellum but not in the hippocampus. In contrast, the phosphorylation of c-Jun was increased only in the hippocampus. These results indicate that ECS can induce the region-specific phosphorylation of MAPK-downstream transcription factors in rat hippocampus and cerebellum. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Electroconvulsive shock; Hippocampus; Cerebellum; ATF-2, Elk-1, c-Jun
Electroconvulsive shock (ECS) induces the expression of the immediate early genes (IEGs) such as c-fos and tetradecanoyl phorbol acetate-inducible sequences (TIS) in the rat brain [4,9]. Previously, we have reported that the induction patterns of IEGs after ECS vary depending on the brain regions. For example, TIS genes were induced more rapidly in the cerebellum than in the hippocampus [9]. Our previous experiments also show that the activation of mitogen activated protein kinases (MAPKs; ERK1/2, JNKs and p38) after ECS is different between the hippocampus and the cerebellum [11,14]. The phosphorylation of ERK1/2, p38 and JNKs was clearly increased after ECS in the hippocampus. However, in the cerebellum, only p38 showed an increased phosphorylation that had intensity comparable to that in the hippocampus. This study was conducted to determine whether ECS could induce the region-specific * Corresponding author. Tel.: 182-2-760-2204; fax: 182-2-7447241. E-mail address: [email protected] (Y.S. Kim).
phosphorylation of MAPK-downstream transcription factors, ATF-2, Elk-1, c-Jun, three important regulatory proteins for the induction of IEGs, in rat hippocampus and cerebellum. The activation of ATF-2 requires the phosphorylation at Thr69 and Thr71, which can be mediated by JNKs and p38 [5,15]. The phosphorylation at Ser383 and Ser389 of Elk-1 is critical for its transcriptional activity and can be accomplished by ERKs, JNKs and p38 [17]. Transcription factor cJun is regarded as a kind of IEGs and its expression is regulated by various stresses in neuronal and non-neuronal cell [13]. The transcriptional activity of c-Jun is regulated by the phosphorylation at Ser63 and Ser73, which is catalyzed by JNKs [13]. Recent data suggest that c-Jun is phosphorylated by other kinases as well, e.g. ERKs [7]. These associations between MAPKs and transcription factors have mostly emerged from cell culture models and it is difficult to extrapolate them directly to the brain in vivo [1,14]. However, very few studies have been conducted concerning the activation of transcription factors following
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ECS in vivo. Bhat et al. [1] reported that the phosphorylation of Elk-1 was increased in the cerebral cortex but not in the hippocampus after ECS. Brecht et al. [2] reported an increase in the nuclear phosphorylated c-Jun after ECS in many brain regions. Nevertheless, these studies [1,2] were about the activation of a single transcription factor and thus did not pursue the regional differences in the activation of signal transduction system. Male Sprague–Dawley rats (150–200 g) were used in this study. Throughout the entire procedure animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Electroconvulsive shock was administered to the animals via ear-clip electrodes (130 V, 0.5 s, Medicraft B-24). Control animals were identically handled except they did not receive any electric current (sham treatment). The rats were decapitated at 0, 1, 2, 5, 10, 30, 60, and 120 min after ECS. The hippocampus and the cerebellum were homogenized in ten volumes (v/w) of pre-chilled buffer containing 25 mM HEPES (pH 7.9), 200 mM NaCl, 1.5 mM MgCl2, 0.2% NP-40, 1 mM DTT, 0.5 mM EDTA, 1 mM PMSF, 20 mM beta-glycerophosphate, 2 mM NaF, 0.1 mM Na3VO4, and 2 mg/ml leupeptin. The Homogenates were centrifuged and the supernatants were boiled with Laemmli’s sample buffer. Immunoblotting was performed using anti-phospho-ATF-2 (Thr71), anti-phospho-Elk-1 (Ser383) and anti-phospho-c-Jun (Ser73) antibodies (New England Biolab.). Signals were detected using ECL system (Pierce). The membranes were then reprobed with anti-ATF-2, anti-Elk-1 and anti-c-Jun antibodies (New England Biolab.) to determine the amounts of the proteins. After confirming the temporal pattern of the phosphorylation, six independent sets of samples were prepared at 2 and 30 min after ECS and statistical analysis was performed. Immunoblot data were processed by the densitometer and the paired t-test was performed on the quantified data. The criterion for significance was P , 0:05. ECS caused a substantial phosphorylation of ATF-2 in both the hippocampus and the cerebellum without altering the protein amount of ATF-2. The phosphorylation started to increase at 1 min and reached peak at about 5 min after ECS. Then it decreased slowly, but was still above the basal level at 120 min after ECS in the hippocampus and the cerebellum (Fig. 1A). At 2 min after ECS, there was a statistically significant increase in the phosphorylation of ATF-2 compared to the sham condition in the hippocampus (P ¼ 0:040) and the cerebellum (P ¼ 0:039) (Fig. 1B). The level of increased phosphorylation in the hippocampus was greater than that in the cerebellum (210.5 ^ 63.6 versus 122.0 ^ 19.4%). At 30 min after ECS, this statistical difference from the sham control was not observed (Fig. 1B). In the cerebellum, ECS increased the phosphorylation of Elk-1 without changing the total protein amount (Fig. 1A). Two minutes after ECS, at which time the phosphorylation of Elk-1 reached peak level, the intensity of phospho-Elk-1 increased by 205% compared to the basal level (P ¼ 004; Fig. 1B). The basal level of Elk-1 phosphorylation was
recovered at 30 min after ECS (Figs. 1A,B). However, neither the protein amount nor the phosphorylation of Elk1 increased in the hippocampus after ECS (Figs. 1A,B). The phosphorylation of c-Jun in the hippocampus began to increase at 1 min, reached peak level at 30–60 min, and did not return to its basal level even at 120 min after ECS (Fig. 1A). In contrast to ATF-2 and Elk-1, the protein amount of c-Jun in the hippocampus was also increased by ECS. The expression of c-Jun protein started to increase at 30 min and continued to increase until 120 min (Fig. 1A). ECS resulted in significant increase of both phosphorylation (174.9 ^ 57.3%, P ¼ 0:043) and protein amount (222.7 ^ 41.5%, P ¼ 0:010) when analyzed at 30 min after ECS (Fig. 1B). Therefore, increased protein amount itself might contribute to the increase in phosphorylation of c-Jun at 30 min after ECS. Whereas, at 2 min after ECS, the phosphorylation of c-Jun (143.2 ^ 36.4%) tended to increase, but did not reach statistical significance (P ¼ 0:056). At these earlier time points, the increased level of phosphorylated c-Jun can not have resulted from the increase in the protein amount, because there was not enough time for protein synthesis (Figs. 1A,B). In contrast to the hippocampus, there was no significant increase in the amount and phosphorylation of c-Jun in the cerebellum (Figs. 1A,B). In this study, ECS induced region-specific phosphorylation of MAPK-downstream transcription factors in rat hippocampus and cerebellum. This finding is supported by the observation that ECS resulted in the phosphorylation of ATF-2 and c-Jun in the hippocampus compared to the phosphorylation of ATF-2 and Elk-1 in the cerebellum. The mechanism leading to the selective phosphorylation of transcription factor is still uncertain, but it is possible that the selective phosphorylation of transcription factors may result from the region-specific activation of MAPKs. The activation of JNKs and p38 leads to the phosphorylation of ATF-2 in cell lines [5,15], and our previous study showed that ECS activated JNKs and p38 in rat hippocampus but activated only p38 in the cerebellum [14]. Therefore, the phosphorylation of ATF-2 in the hippocampus after ECS seems to parallel the activation of JNK and p38. However, in the cerebellum, a slightly increased phosphorylation of ATF-2 after ECS may result from the activation of p38 alone. Although all members of MAPKs were activated in the hippocampus [11,14] and all members of MAPKs phosphorylate Elk-1 in cell culture system [17], the increase in the phosphorylation of Elk-1 was not detected after ECS in the hippocampus. The lack of the phosphorylated Elk-1 in the hippocampus after ECS was also reported by Bhat et al. [1]. They explained that the lack of Elk-1 phosphorylation in the hippocampus might result from the limited localization of the activated ERKs to the cytoplasm, and not the nucleus. But MAPKs other than ERKs (i.e. JNK and p38) known to phosphorylate Elk-1, were also activated in the hippocampus after ECS [14,17] and their downstream transcription factors such as ATF-2 and c-Jun were also phosphorylated
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Fig. 1. The phosphorylation of AFT-2, Elk-1, and c-Jun in rat hippocampus and cerebellum after ECS. (A) The representative blots showing the time course of the phosphorylation of ATF-2, Elk-1 and c-Jun in the hippocampus and cerebellum after ECS. S indicates sham-treated rats that did not receive the electric current. Time (min) indicates the minutes at which the rats were sacrificed after single ECS. (B) The changes in the amount of phosphorylation of ATF-2, Elk-1, c-Jun, and the amount of total c-Jun protein in the hippocampus and cerebellum at 2 and 30 min after ECS. At least six independent experiments were carried out, and the average Relative O.D. (optical densities) was expressed as a percentage of the sham-treated control at ECS 2 or 30 min. The asterisks (*) indicate statistically significant difference compare to sham (P , 0:05, paired t-test).
in the hippocampus in the present study. Therefore the lack of Elk-1 phosphorylation in the hippocampus is not fully explained by the limited localization of ERKs to the cytoplasm. In the cerebellum, ECS increased the phosphorylation of p38 but not of JNK [14], and the increase in the phosphorylation of ERKs in the cerebellum was far less than that in the hippocampus [11]. So, it is plausible that the phosphorylation of Elk-1 in the cerebellum is mainly due to the activation of p38. The differential activation of c-Jun has been suggested by our previous study [14], which showed that the activity of JNKs, major kinases for c-Jun, was dramatically enhanced in a few minutes after ECS in the hippocampus but not in the cerebellum [14]. There have been recent reports of discrepancies between the phosphorylation of c-Jun and the activity of JNKs [7]. But in our observation, the lack of phosphorylation of c-Jun in the cerebellum can be attributed to the lack of JNK activation in this region. Naturally, we should not presume that the phosphorylation of ATF-2, Elk-1 and c-Jun after ECS is
solely determined by MAPKs, for ECS activates multiple signaling pathways that converge in complex manner to regulate the transcription factors. For instance, we have through previous experiments seen the activation of Rafs in the rat brain after ECS [12], and Elk-1 is known to be phosphorylated by a Raf-dependent but MAPK-independent pathway [3]. Just as the phosphorylation of ATF-2, Elk-1 and c-Jun may not be solely determined by MAPKs, previously reported tissue-specific differences in the induction of IEGs after ECS in the rat brain can not be entirely explained by the results in the present study, since the phosphorylation of transcription factors does not always lead to increase in its transcriptional activity [7]. However, the phosphorylations of transcription factors do suggest a spatiotemporal association with the induction of IEGs. And the regional difference in the phosphorylation of transcription factors may be better grasped by observing interactions among many regulatory elements in the promoter region. ECS
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was found to increase the phosphorylation of CREB, another member of ATF/CREB family transcription factors, in the hippocampus but not in the cerebellum after ECS [8], indicating that ECS may induce the IEGs expression in the hippocampus through activation of the CRE motif. c-Jun can also bind to the CRE motifs when forming a heterodimer with ATF-2 [6]. The simultaneous phosphorylation of the c-Jun and ATF-2 was observed in the hippocampus, but not in the cerebellum, suggesting that region-specific phosphorylation of c-Jun and ATF-2 following ECS in the hippocampus may induce, via CRE, region-specific induction of IEGs. Nurr 1, a growth factor-inducible member of the steroid/thyroid hormone receptor gene superfamily, is another gene that contains CRE but not SRE in the promoter region [16]. There was a report of rapid induction of Nurr 1 by single ECS in rat hippocampus but not in other brain regions including the cerebellum [16]. On the other hand, the signal pathways that activate Elk-1 are a major input pathway for SRE-driven IEGs induction [10], and may play important role in induction of IEGs following ECS in the cerebellum but not in the hippocampus. The promoter region of TIS 8 contains five SREs and the SREs are the dominant regulators of TIS 8 expression [6]. In previous study [9], ECS was found to induce many TIS genes both in the cerebellum and the hippocampus, but the induction level of only TIS 8 messenger RNA (mRNA) among TIS gene was much greater in the cerebellum. The expression of TIS 8 is known to be regulated by other transcription factors as well as Elk-1, which might explain the induction of the gene in the hippocampus in the absence of Elk-1 phosphorylation. But the region-specific phosphorylation of Elk-1 might explain the stronger induction of TIS 8 gene in the cerebellum following ECS. In summary, we have shown that ECS can induce the region-specific phosphorylation of MAPKs-downstream transcription factors in rat hippocampus and cerebellum. The results from this study, in conjunction with the previous reports of region specific activation of MAPKs and induction of IEGs after ECS, suggest that there may be regionspecific difference in the activation of intracellular signal transduction pathway after ECS in the rat brain This study was supported by Seoul National University Hospital Research Fund (03-99-70) and in part by 2002 BK21 project for Medicine, Dentistry and Pharmacy.
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