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Region-specific phosphorylation of ERK5–MEF2C in the rat frontal cortex and hippocampus after electroconvulsive shock
Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 749 – 753 www.elsevier.com/locate/pnpbp
Short communication
Region-specific phosphorylation of ERK5–MEF2C in the rat frontal cortex and hippocampus after electroconvulsive shock Se Chang Yoona, Yong Min Ahnb, Sook Ja Junc, Yeni Kimb, Ung Gu Kangb, Joo-Bae Parkd, Yong Sik Kimb,* a
Department of Psychiatry, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, 135-710, Republic of Korea b Department of Psychiatry and Behavioral Science, Institute of Human Behavioral Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea c Clinical Research Institute, Seoul National University Hospital, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Republic of Korea d Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746, Republic of Korea Accepted 6 April 2005 Available online 23 May 2005
Abstract ERK5 – MEF2C has been implicated in many aspects of neuronal survival and neuroprotection. Neurotrophic effects have been considered as one of the mechanisms in therapeutic electroconvulsive shock (ECS). To investigate whether ECS activates ERK5 – MEF2C, we examined the phosphorylation of ERK5, along with its downstream molecule MEF2C, after ECS in the rat frontal cortex and hippocampus. Increased phosphorylation of ERK5 was observed immediately after ECS, but was barely detectable from 2 min after ECS in both the frontal cortex and the hippocampus. The level of MEF2C phosphorylation was decreased immediately after ECS in both regions. It was increased from 2 min and maintained until 10 min after ECS in the frontal cortex, but it returned to the basal level from 2 min after ECS in the hippocampus. Taken together, these results suggest that ECS can regulate the region-specific activity of ERK5 – MEF2C pathways in the rat brain. D 2005 Elsevier Inc. All rights reserved. Keywords: ECS; ERK5; MEF2C; Neuroprotection; Neuronal survival; Signal transduction
1. Introduction Extracellular signal-regulated kinase5 (ERK5), also known as big mitogen-activated kinase 1, is a member of the mitogen-activated protein kinase (MAPK) family. Similar to other MAPKs, ERK5 is activated by various stimuli, such as stress, growth factors and the G-protein coupled receptor ligands (Kato et al., 1998; Marinissen et al., 1999; Yan et al., 1999). MAP kinase kinase, MEK5, has Abbreviations: ECS, electroconvulsive shock; ERK, extracellular signal-regulated kinase; MAPKs, mitogen activated protein kinases; MEF, myocyte enhancer factor. * Corresponding author. Tel.: +82 2 760 2204; fax: +82 2 744 7241. E-mail address: [email protected] (Y.S. Kim). 0278-5846/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2005.04.006
been reported to be specific to ERK5 and full activation of ERK5 requires dual phosphorylation at Thr-219 and Tyr221 residues by this upstream kinase (Kato et al., 1997; Mody et al., 2003). The transcription factor MEF2C is a well-recognized substrate of ERK5, although it can also be a substrate of ERK1/2 (Gille et al., 1995; Kato et al., 1997; Yang et al., 1998). The phosphorylation of Ser-387 by ERK5 is known to increase the transcriptional activity of MEF2C (Kato et al., 1997). ERK5 has been implicated in neurogenesis in early development stage of the cerebral cortex and neuronal survival (Cavanaugh et al., 2001; Liu et al., 2003). MEF2C, the downstream molecule of ERK5, has also been implicated in neuronal survival and synaptic function in the primary neuronal culture (Mao et al., 1999). However, these
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finding are yet to be fully established in the adult central nervous system. ERK5 was reported barely detectable in the cerebral cortex after the postnatal day 49 (Liu et al., 2003), but recently the increased activation of ERK5 has been reported in the adult rat hippocampus after ischemia – reperfusion injury (Wang et al., 2004). We previously reported that the electroconvulsive shock (ECS), an animal model for the electroconvulsive therapy, activates the MAPK family kinases, ERK1/2, p38 and JNK, and their downstream molecules, ATF-2 and Elk-1 (Kang et al., 1994; Oh et al., 1999; Ahn et al., 2002). While membrane depolarization and neuronal growth factors are reported to mediate cellular responses after ECS (Jung et al., 1996; Morgan and Curran, 1991). Therefore, we hypothesized that ECS may also activate ERK5 and its downstream molecule MEF2C. However, our experiences indicate that the activity of MAPK signaling cascade in the brain is not always predictable by extrapolating the results from cell line experiments. Furthermore, the activation of MAPK cascade after ECS also showed regional specificity (Jeon et al., 1998; Oh et al., 1999; Ahn et al., 2002). In order to understand the effect of ECS on the activities of ERK5 and MEF2C in the brain, we investigated the patterns of phosphorylation of ERK5 and MEF2C after ECS in frontal cortex and hippocampus of rat brain.
Santa Cruz Biotechnology). The authors followed the experimental conditions specified by the manufactures. To confirm the specificity of primary antibodies for the phosphorylation status, we compared the molecular sizes with the antibodies which could detect the total form of ERK5 and MEF2C. For phospho-ERK5, because the antibody could also detect the phosphorylated ERK1/2, we confirmed the specificity for the identified phosphorylation status with comparison of the patterns detected by the specific antibodies to phosphorylated ERK1/2 (Thr202/ Tyr204, Cell signaling Technology). All the experiments were repeated at least three times and the results are expressed as percentage of the sham-treated control, which are presented as mean and standard error. Statistical analysis was performed using independent samples t-test. p < 0.05 was considered significant.
2. Methods 2.1. Animal treatments Male Sprague –Dawley rats, ranging from 150 to 200 g, were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council of U.S.A., 1996). The rats were given ECS (130 V, 0.5 s, Medcraft Model B24-III ECT) via ear-clip electrodes. The control animals were treated in the same way as the ECStreated animals but without the electric current (sham treatment). The animals were decapitated at given time points (0, 2, 10, and 30 min after ECS), and the frontal cortex and hippocampus were separated on ice and immediately frozen in liquid nitrogen. The frozen tissues were stored at 70 -C until analysis. 2.2. Tissue preparation and analysis The tissues were homogenized in a glass-Teflon homogenizer in 10 v/w of an ice-cold homogenization buffer, as previously described (Roh et al., 2003). The homogenates, 70 Ag of protein per lane, were separated by 8% SDS-PAGE. Immunoblotting was performed in order to determine the changes in the amount as well as the phosphorylation status of the proteins with antibodies to ERK5 (Cell Signaling Technology), phospho-ERK5 (Thr218/Tyr-220, Cell Signaling Technology), MEF2C (Cell Signaling Technology), and phospho-MEF2C (Ser-387,
Fig. 1. The phosphorylation of ERK5, MEF2C, and ERK1/2 in the rat frontal cortex after ECS. The representative blots show the time course of the phosphorylation and amount of ERK5, MEF2C, and ERK1/2 in the frontal cortex after ECS. Time (min) indicates the minutes at which the rats were sacrificed after a single administration of ECS. S indicates the sham treatment. The arrows indicate each protein band. At least three independent experiments were performed. The density of the immunoblot was analyzed, and the relative optical densities (OD) are percentages of the OD at each time compared to the OD at sham. The average values and standard deviation are presented in the graph. Asterisks (*) indicate statistically significant difference of OD of each time point from the OD at sham ( p < 0.05, independent samples t-test).
S.C. Yoon et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 749 – 753
3. Results 3.1. Phosphorylation of ERK5 – MEF2C in the frontal cortex after ECS In the frontal cortex, the basal level of phospho-ERK5 after sham ECS was very low. The increased phosphorylation of ERK5 was observed immediately (designated 0 min in Fig. 1) after ECS and the increase was more than 2-folds compared to sham ECS (234 T 26% of sham, p = 0.0063, n = 3). From 2 min after ECS, the band of phospho-ERK5 was detectable but the density of the band was comparable to that of sham ECS. Phospho-MEF2C was barely detectable after sham ECS in the frontal cortex. Immediately after ECS, the phosphorylation of MEF2C was decreased compared to sham ECS (35 T 8% of sham, p = 0.0028, n = 3). High increase in the phosphorylation of MEF2C, more than
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2.5-folds greater than that of sham (240 T 76% of sham, p = 0.0429, n = 3), was observed from 2 min after ECS. The increase was maintained at 10 min after ECS (240 T 48% of sham, p = 0.0188, n = 3), and returned to the basal level at 30 min after ECS. As previously reported, ERK1/2 phosphorylation in frontal cortex was increased at 2 min after ECS (Jeon et al., 1998). There was no apparent change in the amount of ERK5 or MEF2C after ECS in our experimental conditions (Fig. 1). 3.2. Phosphorylation of ERK5 –MEF2C in the hippocampus after ECS In the hippocampus, increased phosphorylation of ERK5, at minimum 2.5 folds greater than that of sham ECS (290 T 26% of sham, p = 0.0031, n = 3), was observed immediately after ECS (designated 0 min in Fig. 2). From 2 min after ECS, the band of phospho-ERK5 was detectable, but the density of the band was similar to that of sham ECS. The level of phospho-MEF2C after sham ECS was quite high compared to that of the frontal cortex, indicating that MEF2C was already basally phosphorylated in the hippocampus. However, immediately after ECS the phosphorylation was dramatically reduced compared to sham ECS (8 T 3% of sham, p = 0.0002, n = 3), and returned to the basal level from 2 min after ECS and the basal level was maintained until 30 min after ECS. As previously reported, ERK1/2 phosphorylation in hippocampus was increased at 2 min after ECS (Kang et al., 1994). There was no apparent change in the amount of ERK5 or MEF2C after ECS in our experimental conditions (Fig. 2).
4. Discussion 4.1. ECS increase the phosphorylation of ERK5 after ECS in the rat frontal cortex and hippocampus
Fig. 2. The phosphorylation of ERK5, MEF2C, and ERK1/2 in the rat hippocampus after ECS. The representative blots show the time course of the phosphorylation and amount of ERK5, MEF2C, and ERK1/2 in the hippocampus after ECS. Time (min) indicates the minutes at which the rats were sacrificed after a single administration of ECS. S indicates the sham treatment. The arrows indicate each protein band. At least three independent experiments were performed. The density of the immunoblot was analyzed, and the relative optical densities (OD) are percentages of the OD at each time compared to the OD at sham. The average values and standard errors are presented in the graph. Asterisks (*) indicate statistically significant difference of OD of each time point from the OD at sham ( p < 0.05, independent samples t-test).
The results in this report show that an increase of ERK5 phosphorylation was observed immediately after ECS in the frontal cortex and hippocampus. In addition, the phosphorylation of ERK5 occurred earlier than that of ERK1/2 after ECS in the frontal cortex and hippocampus. It has been reported that ERK5 is activated by neurotrophins but not by membrane depolarization in cerebellar granule cell cultures (Mao et al., 1999). In our ECS paradigm where phasic neuronal depolarization accompanies the seizure activity, ERK5 is activated (phosphorylated) in both frontal cortex and hippocampus. And also in contrast to the reports in which the phosphorylation of ERK5 occurred later than that of ERK1/2 in cortical neurons treated with neurotrophins (Cavanaugh et al., 2001), our results show more prompt activation of ERK5 compared to ERK1/2 after ECS. The discrepancies in these ERK5 activation patterns may reflect differences in the signaling pathways recruited after various
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stimuli and the complexity of the mechanism involved in neuronal survival and neuroprotection. 4.2. Phosphorylation of MEF2C in differential pattern in the rat frontal cortex and the hippocampus The results also showed an increase of MEF2C phosphorylation after ECS, but only in the frontal cortex. The transcriptional activity of MEF2C is stimulated either by membrane depolarization or neurotrophins (Mao et al., 1999), while p38 or ERK5 critically phosphorylates Ser-387 residues of MEF2C (Han et al., 1997; Kato et al., 1997; Mody et al., 2003). We previously reported that the phosphorylation of p38 MAPK is also increased after ECS in the rat hippocampus (Oh et al., 1999), along with phosphorylation of ERK5. These findings led us to hypothesize that MEF2C phosphorylation in the hippocampus would be increased after ECS. However, the results showed that this was not the case. Unexpectedly, MEF2C phosphorylation was reduced immediately after ECS in the frontal cortex and hippocampus. We observed immediate dephosphorylation of various signaling molecules such as the glycogen synthase kinase 3 beta and amphiphysin 2 after ECS (Koo et al., 2002; Roh et al., 2003). Although the mechanism underlying decreases in phosphorylation is still unclear, the decrease of Ser-387-MEF2C immediately after ECS could be explained in a similar manner. Furthermore, it may be possible that the immediate activation of serine phosphatases after ECS may contribute to the lack of increase in phosphorylation in the hippocampus, and that the differences in MEF2C phosphorylation between the cerebral cortex and the hippocampus may have resulted from region specific regulation in the balance between kinases and phosphatases.
5. Conclusion The authors have shown that ECS increase the phosphorylation of ERK5 after immediate ECS in the rat frontal cortex and hippocampus. Also, we showed that the phosphorylation of MEF2C after ECS was increased in the frontal cortex only, along with the unexpected finding that MEF2C phosphorylation was reduced immediately after ECS in the frontal cortex and hippocampus. Taken together, these results suggest that ECS can regulate the region-specific activity of ERK5 – MEF2C pathways in the rat brain.
Acknowledgments This study was supported by grant from the Korea Science and Engineering Foundation (R13-2002-01201001-0), and in part by the BK21 project for Medicine, Dentistry and Pharmacy.
References Ahn, Y.M., Kang, U.G., Oh, S.W., Juhnn, Y.S., Joo, Y.H., Park, J.B., Kim, Y.S., 2002. Region-specific phosphorylation of ATF-2, Elk-1 and c-Jun in rat hippocampus and cerebellum after electroconvulsive shock. Neurosci. Lett. 329, 9 – 12. Cavanaugh, J.E., Ham, J., Hetman, M., Poser, S., Yan, C., Xia, Z., 2001. Differential regulation of mitogen-activated protein kinases ERK1/2 and ERK5 by neurotrophins, neuronal activity, and cAMP in neurons. J. Neurosci. 21, 434 – 443. Gille, H., Kortenjann, M., Thomae, O., Moomaw, C., Slaughter, C., Cobb, M.H., Shaw, P.E., 1995. ERK phosphorylation potentiates Elk-1mediated ternary complex formation and transactivation. EMBO J. 14, 951 – 962. Han, J., Jiang, Y., Li, Z., Kravchenko, V.V., Ulevitch, R.J., 1997. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386, 296 – 299. Jeon, S.H., Yoo, B.H., Kang, U.K., Ahn, Y.M., Bae, C.D., Park, J.B., Kim, Y.S., 1998. MKP-1 induced in rat brain after electroconvulsive shock is independent of regulation of 42- and 44-kDa MAPK activity. Biochem. Biophys. Res. Commun. 249, 692 – 696. Jung, H.Y., Kang, U.G., Ahn, Y.M., Joo, Y.H., Park, J.B., Kim, Y.S., 1996. Induction of tetradecanoyl phorbol acetate-inducible sequence (TIS) genes by electroconvulsive shock in rat brain. Biol. Psychiatry 40, 503 – 507. Kang, U.G., Hong, K.S., Jung, H.Y., Kim, Y.S., Seong, Y.S., Yang, Y.C., Park, J.B., 1994. Activation and tyrosine phosphorylation of 44-kDa mitogen-activated protein kinase (MAPK) induced by electroconvulsive shock in rat hippocampus. J. Neurochem. 63, 1979 – 1982. Kato, Y., Kravchenko, V.V., Tapping, R.I., Han, J., Ulevitch, R.J., Lee, J.D., 1997. BMK1/ERK5 regulates serum-induced early gene expression through transcription factor MEF2C. EMBO J. 16, 7054 – 7066. Kato, Y., Tapping, R.I., Huang, S., Watson, M.H., Ulevitch, R.J., Lee, J.D., 1998. Bmk1/Erk5 is required for cell proliferation induced by epidermal growth factor. Nature 395, 713 – 716. Koo, Y.J., Kim, S.J., Jeon, S.H., Kim, S.R., Kang, U.G., Park, J.B., Kim, Y.S., 2002. Electroconvulsive shock increases the phosphorylation of amphiphysin II in the rat cerebellum. Neurosci. Lett. 330, 135 – 138. Liu, L., Cavanaugh, J.E., Wang, Y., Sakagami, H., Mao, Z., Xia, Z., 2003. ERK5 activation of MEF2-mediated gene expression plays a critical role in BDNF-promoted survival of developing but not mature cortical neurons. Proc. Natl. Acad. Sci. U. S. A. 100, 8532 – 8537. Mao, Z., Bonni, A., Xia, F., Nadal-Vicens, M., Greenberg, M.E., 1999. Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Science 286, 785 – 790. Marinissen, M.J., Chiariello, M., Pallante, M., Gutkind, J.S., 1999. A network of mitogen-activated protein kinases links G protein-coupled receptors to the c-jun promoter: a role for c-Jun NH2-terminal kinase, p38s, and extracellular signal-regulated kinase 5. Mol. Cell. Biol. 19, 4289 – 4301. Mody, N., Campbell, D.G., Morrice, N., Peggie, M., Cohen, P., 2003. An analysis of the phosphorylation and activation of extracellular-signalregulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro. Biochem. J. 372, 567 – 575. Morgan, J.I., Curran, T., 1991. Proto-oncogene transcription factors and epilepsy. Trends Pharmacol. Sci. 12, 343 – 349. National Research Council, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC. Oh, S.W., Ahn, Y.M., Kang, U.G., Kim, Y.S., Park, J.B., 1999. Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock. Neurosci. Lett. 271, 101 – 104. Roh, M.S., Kang, U.G., Shin, S.Y., Lee, Y.H., Jung, H.Y., Juhnn, Y.S., Kim, Y.S., 2003. Biphasic changes in the Ser-9 phosphorylation of glycogen synthase kinase-3beta after electroconvulsive shock in the rat brain. Prog. Neuro-psychopharmacol. Biol. Psychiatry 27, 1 – 5.
S.C. Yoon et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 749 – 753 Wang, R.M., Zhang, Q.G., Zhang, G.Y., 2004. Activation of ERK5 is mediated by N-methyl-d-aspartate receptor and L-type voltage-gated calcium channel via Src involving oxidative stress after cerebral ischemia in rat hippocampus. Neurosci. Lett. 357, 13 – 16. Yan, C., Takahashi, M., Okuda, M., Lee, J.D., Berk, B.C., 1999. Fluid shear stress stimulates big mitogen-activated protein kinase 1 (BMK1)
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Short communication
Region-specific phosphorylation of ERK5–MEF2C in the rat frontal cortex and hippocampus after electroconvulsive shock Se Chang Yoona, Yong Min Ahnb, Sook Ja Junc, Yeni Kimb, Ung Gu Kangb, Joo-Bae Parkd, Yong Sik Kimb,* a
Department of Psychiatry, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, 135-710, Republic of Korea b Department of Psychiatry and Behavioral Science, Institute of Human Behavioral Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea c Clinical Research Institute, Seoul National University Hospital, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Republic of Korea d Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746, Republic of Korea Accepted 6 April 2005 Available online 23 May 2005
Abstract ERK5 – MEF2C has been implicated in many aspects of neuronal survival and neuroprotection. Neurotrophic effects have been considered as one of the mechanisms in therapeutic electroconvulsive shock (ECS). To investigate whether ECS activates ERK5 – MEF2C, we examined the phosphorylation of ERK5, along with its downstream molecule MEF2C, after ECS in the rat frontal cortex and hippocampus. Increased phosphorylation of ERK5 was observed immediately after ECS, but was barely detectable from 2 min after ECS in both the frontal cortex and the hippocampus. The level of MEF2C phosphorylation was decreased immediately after ECS in both regions. It was increased from 2 min and maintained until 10 min after ECS in the frontal cortex, but it returned to the basal level from 2 min after ECS in the hippocampus. Taken together, these results suggest that ECS can regulate the region-specific activity of ERK5 – MEF2C pathways in the rat brain. D 2005 Elsevier Inc. All rights reserved. Keywords: ECS; ERK5; MEF2C; Neuroprotection; Neuronal survival; Signal transduction
1. Introduction Extracellular signal-regulated kinase5 (ERK5), also known as big mitogen-activated kinase 1, is a member of the mitogen-activated protein kinase (MAPK) family. Similar to other MAPKs, ERK5 is activated by various stimuli, such as stress, growth factors and the G-protein coupled receptor ligands (Kato et al., 1998; Marinissen et al., 1999; Yan et al., 1999). MAP kinase kinase, MEK5, has Abbreviations: ECS, electroconvulsive shock; ERK, extracellular signal-regulated kinase; MAPKs, mitogen activated protein kinases; MEF, myocyte enhancer factor. * Corresponding author. Tel.: +82 2 760 2204; fax: +82 2 744 7241. E-mail address: [email protected] (Y.S. Kim). 0278-5846/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2005.04.006
been reported to be specific to ERK5 and full activation of ERK5 requires dual phosphorylation at Thr-219 and Tyr221 residues by this upstream kinase (Kato et al., 1997; Mody et al., 2003). The transcription factor MEF2C is a well-recognized substrate of ERK5, although it can also be a substrate of ERK1/2 (Gille et al., 1995; Kato et al., 1997; Yang et al., 1998). The phosphorylation of Ser-387 by ERK5 is known to increase the transcriptional activity of MEF2C (Kato et al., 1997). ERK5 has been implicated in neurogenesis in early development stage of the cerebral cortex and neuronal survival (Cavanaugh et al., 2001; Liu et al., 2003). MEF2C, the downstream molecule of ERK5, has also been implicated in neuronal survival and synaptic function in the primary neuronal culture (Mao et al., 1999). However, these
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finding are yet to be fully established in the adult central nervous system. ERK5 was reported barely detectable in the cerebral cortex after the postnatal day 49 (Liu et al., 2003), but recently the increased activation of ERK5 has been reported in the adult rat hippocampus after ischemia – reperfusion injury (Wang et al., 2004). We previously reported that the electroconvulsive shock (ECS), an animal model for the electroconvulsive therapy, activates the MAPK family kinases, ERK1/2, p38 and JNK, and their downstream molecules, ATF-2 and Elk-1 (Kang et al., 1994; Oh et al., 1999; Ahn et al., 2002). While membrane depolarization and neuronal growth factors are reported to mediate cellular responses after ECS (Jung et al., 1996; Morgan and Curran, 1991). Therefore, we hypothesized that ECS may also activate ERK5 and its downstream molecule MEF2C. However, our experiences indicate that the activity of MAPK signaling cascade in the brain is not always predictable by extrapolating the results from cell line experiments. Furthermore, the activation of MAPK cascade after ECS also showed regional specificity (Jeon et al., 1998; Oh et al., 1999; Ahn et al., 2002). In order to understand the effect of ECS on the activities of ERK5 and MEF2C in the brain, we investigated the patterns of phosphorylation of ERK5 and MEF2C after ECS in frontal cortex and hippocampus of rat brain.
Santa Cruz Biotechnology). The authors followed the experimental conditions specified by the manufactures. To confirm the specificity of primary antibodies for the phosphorylation status, we compared the molecular sizes with the antibodies which could detect the total form of ERK5 and MEF2C. For phospho-ERK5, because the antibody could also detect the phosphorylated ERK1/2, we confirmed the specificity for the identified phosphorylation status with comparison of the patterns detected by the specific antibodies to phosphorylated ERK1/2 (Thr202/ Tyr204, Cell signaling Technology). All the experiments were repeated at least three times and the results are expressed as percentage of the sham-treated control, which are presented as mean and standard error. Statistical analysis was performed using independent samples t-test. p < 0.05 was considered significant.
2. Methods 2.1. Animal treatments Male Sprague –Dawley rats, ranging from 150 to 200 g, were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council of U.S.A., 1996). The rats were given ECS (130 V, 0.5 s, Medcraft Model B24-III ECT) via ear-clip electrodes. The control animals were treated in the same way as the ECStreated animals but without the electric current (sham treatment). The animals were decapitated at given time points (0, 2, 10, and 30 min after ECS), and the frontal cortex and hippocampus were separated on ice and immediately frozen in liquid nitrogen. The frozen tissues were stored at 70 -C until analysis. 2.2. Tissue preparation and analysis The tissues were homogenized in a glass-Teflon homogenizer in 10 v/w of an ice-cold homogenization buffer, as previously described (Roh et al., 2003). The homogenates, 70 Ag of protein per lane, were separated by 8% SDS-PAGE. Immunoblotting was performed in order to determine the changes in the amount as well as the phosphorylation status of the proteins with antibodies to ERK5 (Cell Signaling Technology), phospho-ERK5 (Thr218/Tyr-220, Cell Signaling Technology), MEF2C (Cell Signaling Technology), and phospho-MEF2C (Ser-387,
Fig. 1. The phosphorylation of ERK5, MEF2C, and ERK1/2 in the rat frontal cortex after ECS. The representative blots show the time course of the phosphorylation and amount of ERK5, MEF2C, and ERK1/2 in the frontal cortex after ECS. Time (min) indicates the minutes at which the rats were sacrificed after a single administration of ECS. S indicates the sham treatment. The arrows indicate each protein band. At least three independent experiments were performed. The density of the immunoblot was analyzed, and the relative optical densities (OD) are percentages of the OD at each time compared to the OD at sham. The average values and standard deviation are presented in the graph. Asterisks (*) indicate statistically significant difference of OD of each time point from the OD at sham ( p < 0.05, independent samples t-test).
S.C. Yoon et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 749 – 753
3. Results 3.1. Phosphorylation of ERK5 – MEF2C in the frontal cortex after ECS In the frontal cortex, the basal level of phospho-ERK5 after sham ECS was very low. The increased phosphorylation of ERK5 was observed immediately (designated 0 min in Fig. 1) after ECS and the increase was more than 2-folds compared to sham ECS (234 T 26% of sham, p = 0.0063, n = 3). From 2 min after ECS, the band of phospho-ERK5 was detectable but the density of the band was comparable to that of sham ECS. Phospho-MEF2C was barely detectable after sham ECS in the frontal cortex. Immediately after ECS, the phosphorylation of MEF2C was decreased compared to sham ECS (35 T 8% of sham, p = 0.0028, n = 3). High increase in the phosphorylation of MEF2C, more than
751
2.5-folds greater than that of sham (240 T 76% of sham, p = 0.0429, n = 3), was observed from 2 min after ECS. The increase was maintained at 10 min after ECS (240 T 48% of sham, p = 0.0188, n = 3), and returned to the basal level at 30 min after ECS. As previously reported, ERK1/2 phosphorylation in frontal cortex was increased at 2 min after ECS (Jeon et al., 1998). There was no apparent change in the amount of ERK5 or MEF2C after ECS in our experimental conditions (Fig. 1). 3.2. Phosphorylation of ERK5 –MEF2C in the hippocampus after ECS In the hippocampus, increased phosphorylation of ERK5, at minimum 2.5 folds greater than that of sham ECS (290 T 26% of sham, p = 0.0031, n = 3), was observed immediately after ECS (designated 0 min in Fig. 2). From 2 min after ECS, the band of phospho-ERK5 was detectable, but the density of the band was similar to that of sham ECS. The level of phospho-MEF2C after sham ECS was quite high compared to that of the frontal cortex, indicating that MEF2C was already basally phosphorylated in the hippocampus. However, immediately after ECS the phosphorylation was dramatically reduced compared to sham ECS (8 T 3% of sham, p = 0.0002, n = 3), and returned to the basal level from 2 min after ECS and the basal level was maintained until 30 min after ECS. As previously reported, ERK1/2 phosphorylation in hippocampus was increased at 2 min after ECS (Kang et al., 1994). There was no apparent change in the amount of ERK5 or MEF2C after ECS in our experimental conditions (Fig. 2).
4. Discussion 4.1. ECS increase the phosphorylation of ERK5 after ECS in the rat frontal cortex and hippocampus
Fig. 2. The phosphorylation of ERK5, MEF2C, and ERK1/2 in the rat hippocampus after ECS. The representative blots show the time course of the phosphorylation and amount of ERK5, MEF2C, and ERK1/2 in the hippocampus after ECS. Time (min) indicates the minutes at which the rats were sacrificed after a single administration of ECS. S indicates the sham treatment. The arrows indicate each protein band. At least three independent experiments were performed. The density of the immunoblot was analyzed, and the relative optical densities (OD) are percentages of the OD at each time compared to the OD at sham. The average values and standard errors are presented in the graph. Asterisks (*) indicate statistically significant difference of OD of each time point from the OD at sham ( p < 0.05, independent samples t-test).
The results in this report show that an increase of ERK5 phosphorylation was observed immediately after ECS in the frontal cortex and hippocampus. In addition, the phosphorylation of ERK5 occurred earlier than that of ERK1/2 after ECS in the frontal cortex and hippocampus. It has been reported that ERK5 is activated by neurotrophins but not by membrane depolarization in cerebellar granule cell cultures (Mao et al., 1999). In our ECS paradigm where phasic neuronal depolarization accompanies the seizure activity, ERK5 is activated (phosphorylated) in both frontal cortex and hippocampus. And also in contrast to the reports in which the phosphorylation of ERK5 occurred later than that of ERK1/2 in cortical neurons treated with neurotrophins (Cavanaugh et al., 2001), our results show more prompt activation of ERK5 compared to ERK1/2 after ECS. The discrepancies in these ERK5 activation patterns may reflect differences in the signaling pathways recruited after various
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stimuli and the complexity of the mechanism involved in neuronal survival and neuroprotection. 4.2. Phosphorylation of MEF2C in differential pattern in the rat frontal cortex and the hippocampus The results also showed an increase of MEF2C phosphorylation after ECS, but only in the frontal cortex. The transcriptional activity of MEF2C is stimulated either by membrane depolarization or neurotrophins (Mao et al., 1999), while p38 or ERK5 critically phosphorylates Ser-387 residues of MEF2C (Han et al., 1997; Kato et al., 1997; Mody et al., 2003). We previously reported that the phosphorylation of p38 MAPK is also increased after ECS in the rat hippocampus (Oh et al., 1999), along with phosphorylation of ERK5. These findings led us to hypothesize that MEF2C phosphorylation in the hippocampus would be increased after ECS. However, the results showed that this was not the case. Unexpectedly, MEF2C phosphorylation was reduced immediately after ECS in the frontal cortex and hippocampus. We observed immediate dephosphorylation of various signaling molecules such as the glycogen synthase kinase 3 beta and amphiphysin 2 after ECS (Koo et al., 2002; Roh et al., 2003). Although the mechanism underlying decreases in phosphorylation is still unclear, the decrease of Ser-387-MEF2C immediately after ECS could be explained in a similar manner. Furthermore, it may be possible that the immediate activation of serine phosphatases after ECS may contribute to the lack of increase in phosphorylation in the hippocampus, and that the differences in MEF2C phosphorylation between the cerebral cortex and the hippocampus may have resulted from region specific regulation in the balance between kinases and phosphatases.
5. Conclusion The authors have shown that ECS increase the phosphorylation of ERK5 after immediate ECS in the rat frontal cortex and hippocampus. Also, we showed that the phosphorylation of MEF2C after ECS was increased in the frontal cortex only, along with the unexpected finding that MEF2C phosphorylation was reduced immediately after ECS in the frontal cortex and hippocampus. Taken together, these results suggest that ECS can regulate the region-specific activity of ERK5 – MEF2C pathways in the rat brain.
Acknowledgments This study was supported by grant from the Korea Science and Engineering Foundation (R13-2002-01201001-0), and in part by the BK21 project for Medicine, Dentistry and Pharmacy.
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