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2 inhibition increases dopamine release from differentiated PC12 cells
Neuroscience Letters 684 (2018) 6–12
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
ERK1/2 inhibition increases dopamine release from differentiated PC12 cells D.V. Zosen1, N.A. Dorofeeva, E.V. Chernigovskaya, V.T. Bachteeva, M.V. Glazova
T
⁎
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez pr., 194223 St. Petersburg, Russia
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: PC12 cells Dopamine ERK1/2 U0126 PKG PKA Synapsin I SNAP-25
The release of dopamine (DA) is one of the main steps in the control of neuronal functioning and all CNS. It was demonstrated that many factors such as protein kinases and synaptic proteins are tightly involved in the regulation of DA secretion, but the data are contradictory. Here we analysed an effect of ERK1/2 inhibition on DA secretion from differentiated PC12 cells and evaluated the correlation between the activity of kinases/synaptic proteins and the level of released DA. PC12 cells were differentiated by NGF for 6 days. On the 7th day the cells were incubated for 1, 2 and 4 h with 10μM U0126. Obtained data demonstrated a significant accumulation of DA in the media after 4 h incubation with U0126 that accompanied with upregulation of PKG activity. Analysis of exocytosis proteins demonstrated decreased phosphorylation level of synapsin I and content of SNAP25. Taken together our data proposed an inhibitory role of ERK1/2 in the regulation of catecholamine secretion and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from the cells.
⁎
Corresponding author. E-mail address: [email protected] (M.V. Glazova). Present address: Biocenter Oulu, Laboratory of Developmental Biology, Department of Medical Biochemistry and Molecular Biology, University of Oulu, P.O. Box 5000, Aapistie 5, FIN-90014, Oulu, Finland. 1
https://doi.org/10.1016/j.neulet.2018.06.056 Received 14 March 2018; Received in revised form 25 June 2018; Accepted 28 June 2018 Available online 30 June 2018 0304-3940/ © 2018 Elsevier B.V. All rights reserved.
Neuroscience Letters 684 (2018) 6–12
D.V. Zosen et al.
1. Introduction
2.3. Western blot analysis
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) participate in the regulation of many cellular functions that include proliferation, differentiation, protein synthesis, gene transcription etc. Among ERK1/ 2 substrates tyrosine hydroxylase (TH) is the main rate-limiting enzyme of catecholamine synthesis. ERK1/2 phosphorylates TH at Ser31 and increases enzymatic activity of TH up to two fold. Such a not very impressive increase in enzymatic activity is indeed critical for the regulation of TH basal activity [24]. However, the main regulator of TH activity is cAMP-dependent protein kinase (PKA) that phosphorylates it at Ser40 and activates it up to 15th fold. Other kinases such as Calcium/ calmodulin-stimulated protein kinase II (CaMKII), cyclin-dependent kinase 5 (Cdk5) and cGMP-dependent protein kinase (PKG) also regulate TH activity by phosphorylation at different sites [24]. All these events lead to upregulation of dopamine (DA) synthesis and its accumulation in the neurons that should finalize with DA secretion at the synaptic clefts. However, the mechanisms that regulate catecholamine release are not clearly understood. Many factors could affect on DA release, among these the first discovered and more detailed described are depolarization and intracellular Ca2+ concentration [8]. Study of neurochemical events demonstrated a stimulatory effect of cAMP on catecholamine release [19] and association of PKG activation with upregulation of DA extracellular content [15]. But it is even less known about the participation of ERK1/2 in the regulation of catecholamine release. However, the role of ERK1/2 in the regulation of exocytosis machinery is extensively studied. It was demonstrated that ERK1/2 phosphorylates some cytoskeletal proteins thus participating in exocytosis and synaptic plasticity [25]. To analyze a role of ERK1/2 in DA release we used differentiated PC12 cells. These cells have strong similarity in the synaptic machinery with neurons in the peripheral and central nervous systems that positioned PC12 cells as a perfect model for studding of neurosecretion [26,27]. ERK1/2 is activated by dual-specificity MAP/ERK kinases 1and 2 (MEK1/2), and is the only known substrate for MEK1/2 [22]. Our data demonstrated that treatment with a selective inhibitor of MEK1/2 U0126, which subsequently inhibits ERK1/2, potentiated secretion of DA from cells. Analysis of neurochemical events in the cells showed that inhibition of ERK1/2 was associated with an increase in the activity of cGMP-dependent protein kinase (PKG), but the levels of PKA and CaMKII kinase activity were not affected. Obtained data supposed that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from differentiated mature neurons.
The cells were homogenized in SDS-stop buffer containing 3% betamercaptoethanol and denaturated at 95 °C for 5 min, and when separated on 10% Acrylamide/Bis-Acrylamide gel. The proteins on the gels were transferred to nitrocellulose membranes, which were then incubated overnight with antibody against: TH (#T1299, Sigma), p-TH (Ser31, #AB5423, Millipore), ERK1/2 (#9102, Cell Signaling), pERK1/2 (#4376, Cell Signaling), synapsin I (#AB1543 P, Millipore), psynapsin I (Ser 62/67, #AB9848, Millipore), SNAP25 (#MAB331, Chemicon), VAMP-2 (#104221, Synaptic System), p-CaMKII (Thr286, #MA 1-047, Thermo Scientific), p-VASP Ser239 (#sc-101439, Santa Cruze Biotech.) or p-VASP Ser157 (#sc-101440, Santa Cruze Biotech.) followed by incubation with anti-rabbit or anti-mouse HRP secondary antibody and final chemilumeniscent detection by Super Signal West Dura Extended Duration Substrate (#34075, Thermo Scientific). 2.4. Statistics Statistical analysis was carried out by the Student’s t-test, and values are expressed as mean ± SE for ELISA, and for western blot analysis. p < 0.05 was taken as levels of statistical significance. 3. Results 3.1. Analysis of DA concentration Firstly, we determined DA concentration in the media after 1, 2 and 4 h’ incubation with U0126. Obtained results demonstrated the increasing level of DA in 4 h’ incubation of differentiated PC12 cells with U0126 (Fig. 1) that proposed upregulation of DA release from the cells. 3.2. Effects of U0126 on ERK1/2 and TH phosphorylation After application of U0126 phosphorylation of ERK1/2 was completely disappeared (Fig. 2A). Then we analyzed phosphorylation level of TH at Ser31, which is specific site mainly for ERK1/2 [11]. The results showed significantly decreased phosphorylation of TH at Ser31 at all-time points (Fig. 2B) that revealed catecholamine down-regulated basal secretion. We also obtained an interesting result that in control groups ERK1/2 phosphorylation levels were upregulated in a time-dependent manner (Fig. 2A) and the same changes ware estimated for TH phosphorylation (Fig. 2B), supposing some compensatory mechanism on short-term NGF omission.
2. Material and methods 2.1. Cell culture PC12 cells were incubated on collagen-coated plates (collagen rat tail #11 179 179 001, Roche) in DMEM supplemented with 10% horse serum, 5% FBS, penicillin- streptomycin (all from Sigma). When the cell confluence reached about 50–60% the medium was changed to DMEM supplemented with 1% horse serum and NGF (50 ng/ml, #01-125, Millipore) for stimulation of differentiation. The cells were incubated with NGF for 6 days. On the next day the medium was changed for DMEM only and 10 μM U0126 (#1144, Tocris) was added for 1, 2 and 4 h (n = 6 for each time point). At each time point, media was collected for further ELISA analysis, the cells were homogenized for Western blotting. 2.2. ELISA
Fig. 1. ERK1/2 inhibition stimulates DA release from differentiated PC12 cells. DA concentration (ng/ml) in media after incubation the cells with U0126 during 1, 2 and 4 h. Data are shown as means ± SE. * p < 0.05 versus corresponding control.
DA concentrations in the media were determined by DA ELISA kit (#40-371-25013, GenWay Biothech Inc., San Diego, CA, USA) according to the manufacturer instruction. 7
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Fig. 2. Analysis the phosphorylation levels of ERK1/2 and TH after U0126 treatments. PC12 cells were incubated for 1, 2 and 4 h with U0126 and phosphorylation of ERK1/2 (A) and TH at Ser31 (B) were estimated by Western blot. Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. * p < 0.05 versus corresponding control.
phosphorylation levels was detected in both control and U0126 treated groups (Fig. 3B) in comparison with the groups after 1 h incubation. On the other hand, phosphorylation level of VASP at Ser239 was increased after application of U0126 (Fig. 3C) that proposed upregulation of PKG activity.
3.3. Activity of PKA, PKG and CaMKII We analyzed of PKA, PKG and CaMKII activity by estimation of its phosphorylation levels. Obtained results of CaMKII phosphorylation level at Thr286, which corresponds to CaMKII activation, demonstrated that inhibition of ERK1/2 did not affect it (Fig. 3A). The activity of PKA and PKG was analyzed by estimation of phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) at Ser157 and Ser239, which are specific phosphorylation sites for PKA and PKG correspondently [3]. Expression of VASP protein was demonstrated in many cell types including neurons and PC12 cells where this protein plays a significant role in the regulation the neurite growth cone formation [10]. Our results demonstrated that phosphorylation level of VASP at Ser157 was not affected by U0126 treatment (Fig. 3B) displaying unchanged activity of PKA in comparison with corresponding control groups. However, after 2 and 4 h, significant reduction of VASP Ser157
3.4. Analysis of exocytosis proteins We studied synapsin I protein expression and phosphorylation levels of synapsin I at Ser 62/67, which, like TH, are also the targets for ERK1/2 [4]. While pan synapsin I level was not altered by decreased activity of ERK1/2, the level of synapsin I phosphorylation was significantly decreased (Fig. 4B). Then we analyzed synaptosomal-associated protein 25 (SNAP-25) and vesicle-associated membrane protein 2 (VAMP2) proteins, which belong to SNARE complex. Obtained data demonstrated unchanged protein level of VAMP2 in the cells (data not 8
Neuroscience Letters 684 (2018) 6–12
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Fig. 3. Analysis of the activity of CaMKII, PKA and PKC kinases in differentiated PC12 cells after inhibition of ERK1/2 activity. A – phosphorylation of CaMKII was not changed in the cells after inhibition of ERK1/2 activity. B, C – analysis of PKA and PKC activity after inhibition of ERK1/2 were estimated by phosphorylation levels of VASP at Ser157 (phosphorylation site for PKA) (B), and at Ser 239 (phosphorylation site for PKG) (C). Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. * p < 0.05; ** p < 0.01 versus corresponding control. 9
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Fig. 4. U0126 treatment significantly decreases SNAP-25 expression and phosphorylation of synapsin I. A – expression SNAP-25 protein in the differentiated PC12 cells after incubation with U0126 during 1, 2 and 4 h. B – phosphorylation level of synapsin I at Ser62/67 estimated by Western Blot. Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. * p < 0.05; ** p < 0.01 versus corresponding control.
neurons after inhibition of ERK1/2 by U0126. Thus the authors proposed the inhibitory role of ERK1/2 in the regulation of neurotransmitter release. These data are in line with our observation and confirmed that ERK1/2 repress unstimulated basal secretion of DA in differentiated PC12 cells. We also could not exclude the possibility that accumulation of DA in the media may be caused by inhibition of DA reuptake. Published data demonstrated that an inhibition of ERK1/2 prevents DA uptake in in vitro experiments on isolated synaptosomes [17]. However, Lin with co-authors did not show any effects of U0126 on DA re-uptake in DAT transfected cells [16]. It is known that cocaine blocks DA re-uptake in PC12 cells [1,28], but on the other hand cocaine also significantly increases activity of ERK1/2 [12,14]. So it seems that ERK1/2-dependent regulation of DA re-uptake could be dependent from the cell types or cell environment since in synaptosomes ERK1/2 blockade inhibits DA re-uptake, while cocaine, which activates ERK1/2,
shown). However, the level of SNAP-25 protein was significantly decreased (Fig. 4A). 4. Discussion Here we analyzed a role of ERK1/2 kinase in the regulation of DA release from differentiated PC12 cells. Analysis of DA concentration in the media after inhibition of ERK1/2 demonstrated the increasing level of DA in 4 h’ incubation of differentiated PC12 cells that proposed upregulation of DA release from the cells. In spite of plenty published data [2,5,6,20,21] demonstrating a positive effect of ERK1/2 in the regulation of induced catecholamine secretion our data revealed that inhibition of ERK1/2 activity potentiated the release of DA from differentiated PC12 cells. Recently Subramanian and Morozov [23] also revealed a stimulation of vesicular exocytosis in primary hippocampal 10
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Conflict of interest
has got an inhibitory effect on DA re-uptake too. Summarizing published data, we could not exclude effect of ERK1/2 inhibition on DA reuptake in differentiated PC12 cells. According the arising complexity of these process and difficulties to predict the effect, this question should be addressed to further studies. Then we analyzed ERK-dependent activity of TH, which is the main rate-limiting enzyme of catecholamine synthesis. After U0126 treatments the phosphorylation level of TH at Ser31 was decreased. Such significant elimination of TH phosphorylation at Ser31 could be dependent on ERK1/2 inactivation [24]. However, we did not see complete dephosphorylation of TH as we observed for ERK1/2 that can be explained by the fact of TH phosphorylation at Ser31 also by Cdk5 [24]. We also found an interesting fact that in control groups ERK1/2 phosphorylation levels were upregulated in a time-dependent manner and the same changes were estimated for TH phosphorylation, supposing some compensatory mechanism on short-term NGF omission. Moreover, our data demonstrated that decreased ERK-dependent activity of TH and respectively decreased basal synthesis of DA did not affect the fast release of DA from the cells. It is known that exocytosis of neurotransmitters could be dependent on the activity of PKA, PKG and CaMKII protein kinases. Obtained results demonstrated that inhibition of ERK1/2 did not affect on CaMKII activity level as well as on the activity of PKA. However, our data revealed activation of PKG. PKG highly expresses in the brain and plays a significant role in the regulation of neurotransmitter release, in particular, DA secretion [24]. It would also need to mention that in control group after 4 h’ incubation an increased activity of PKG correlated with the enhanced level of DA in comparison with the 1-hour control group after. Exocytosis machinery is operated by many exocytosis related proteins such as synapsins and soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins. We analyzed if the inhibition of ERK1/2 correlated with some alteration in the exocytosis proteins therefor promoting DA release. Initially we studied synapsin I protein expression and phosphorylation levels of synapsin I at Ser 62/ 67, which, like TH, are also the targets for ERK1/2 [4]. While pan synapsin I level was not altered by decreased activity of ERK1/2, the level of synapsin I phosphorylation was significantly decreased. It is known that synapsins regulate neurotransmitter release. However, its role could be variable. ERK1/2-dependent synapsin I phosphorylation positively regulates glutamate release [4], while Kile with co-authors demonstrated that synapsins play an inhibitory role in DA secretion in vivo and in vitro [13]. Our data confirmed this observation since ERK1/2 dependent inhibition of synapsin I correlated with increased DA secretion. Then we analyzed synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) proteins, which belong to SNARE complex. Obtained data demonstrated unchanged protein level of VAMP2 in the cells (data not shown). However, the level of SNAP-25 protein was significantly decreased. These data corresponds to our previous data, which demonstrated a correlation of SNAP-25 protein level with the level of vasopressin [18] and glutamate release [9]. Moreover, Eisenhofer and co-authors also revealed the same correlation between catecholamine release and expression of SNAP-25 [7].
The authors declare that they have no conflict of interest. Acknowledgments This work was supported by FASO of Russia (#АААА-А18118012290371-3). Part of the analysis was done at Research Resource Center #441590 at Sechenov Institute of Evolutionary Physiology and Biochemistry. References [1] R.B. Badisa, C.S. Batton, E. Mazzio, S.C. Grant, C.B. Goodman, Identification of biochemical and cytotoxic markers in cocaine treated PC12 cells, Sci. Rep. 8 (2018) 2710. [2] E. Bloch-Shilderman, H. Jiang, S. Abu-Raya, M. Linial, P. Lazarovici, Involvement of extracellular signal-regulated kinase (ERK) in pardaxin-induced dopamine release from PC12 cells, J. Pharmacol. Exp. Ther. 296 (2001) 704–711. [3] E. Butt, K. Abel, M. Krieger, D. Palm, V. Hoppe, J. Hoppe, U. Walter, cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets, J. Biol. Chem. 269 (1994) 14509–14517. [4] F. Cesca, P. Baldelli, F. Valtorta, F. Benfenati, The synapsins: key actors of synapse function and plasticity, Prog. Neurobiol. 91 (2010) 313–348. [5] M.E. Cox, C.M. Ely, A.D. Catling, M.J. Weber, S.J. Parsons, Tyrosine kinases are required for catecholamine secretion and mitogen-activated protein kinase activation in bovine adrenal chromaffin cells, J. Neurochem. 66 (1996) 1103–1112. [6] M.E. Cox, S.J. Parsons, Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells, J. Neurochem. 69 (1997) 1119–1130. [7] G. Eisenhofer, T.T. Huynh, A. Elkahloun, J.C. Morris, G. Bratslavsky, W.M. Linehan, Z. Zhuang, B.M. Balgley, C.S. Lee, M. Mannelli, J.W. Lenders, S.R. Bornstein, K. Pacak, Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma, Am. J. Physiol. Endocrinol. Metab. 295 (2008) E1223–1233. [8] A.G. Garcia, A.M. Garcia-De-Diego, L. Gandia, R. Borges, J. Garcia-Sancho, Calcium signaling and exocytosis in adrenal chromaffin cells, Physiol. Rev. 86 (2006) 1093–1131. [9] M.V. Glazova, L.S. Nikitina, K.A. Hudik, O.D. Kirillova, N.A. Dorofeeva, A.A. Korotkov, E.V. Chernigovskaya, Inhibition of ERK1/2 signaling prevents epileptiform behavior in rats prone to audiogenic seizures, J. Neurochem. 132 (2015) 218–229. [10] D.J. Goldberg, M.S. Foley, D. Tang, P.W. Grabham, Recruitment of the Arp2/3 complex and mena for the stimulation of actin polymerization in growth cones by nerve growth factor, J. Neurosci. Res. 60 (2000) 458–467. [11] J.W. Haycock, N.G. Ahn, M.H. Cobb, E.G. Krebs, ERK1 and ERK2, two microtubuleassociated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 2365–2369. [12] G. Kharkwal, D. Radl, R. Lewis, E. Borrelli, Dopamine D2 receptors in striatal output neurons enable the psychomotor effects of cocaine, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 11609–11614. [13] B.M. Kile, T.S. Guillot, B.J. Venton, W.C. Wetsel, G.J. Augustine, R.M. Wightman, Synapsins differentially control dopamine and serotonin release, J. Neurosci. 30 (2010) 9762–9770. [14] A. Lakatos, S. Prinster, A. Vicentic, R.A. Hall, M.J. Kuhar, Cocaine- and amphetamine-regulated transcript (CART) peptide activates the extracellular signal-regulated kinase (ERK) pathway in AtT20 cells via putative G-protein coupled receptors, Neurosci. Lett. 384 (2005) 198–202. [15] D.K. Lee, J.H. Oh, Y.B. Shim, E.S. Choe, Protein kinase G regulates dopamine release, DeltaFosB expression, and locomotor activity after repeated cocaine administration: involvement of dopamine D2 receptors, Neurochem. Res. 38 (2013) 1424–1433. [16] Z. Lin, P.W. Zhang, X. Zhu, J.M. Melgari, R. Huff, R.L. Spieldoch, G.R. Uhl, Phosphatidylinositol 3-kinase, protein kinase C, and MEK1/2 kinase regulation of dopamine transporters (DAT) require N-terminal DAT phosphoacceptor sites, J. Biol. Chem. 278 (2003) 20162–20170. [17] J.A. Moron, I. Zakharova, J.V. Ferrer, G.A. Merrill, B. Hope, E.M. Lafer, Z.C. Lin, J.B. Wang, J.A. Javitch, A. Galli, T.S. Shippenberg, Mitogen-activated protein kinase regulates dopamine transporter surface expression and dopamine transport capacity, J. Neurosci. 23 (2003) 8480–8488. [18] L.S. Nikitina, N.A. Dorofeeva, O.D. Kirillova, A.A. Korotkov, M. Glazova, E.V. Chernigovskaya, Role of the ERK signaling pathway in regulating vasopressin secretion in dehydrated rats, Biotech. Histochem. 89 (2014) 199–208. [19] L. Ribeiro, I. Azevedo, F. Martel, Comparison of the effect of cyclic AMP on the content and release of dopamine and 1-methyl-4-phenylpyridinium (Mpp+) in PC12 cells, Auton Autacoid Pharmacol. 22 (2002) 277–289. [20] J. Rosmaninho-Salgado, I.M. Araujo, A.R. Alvaro, E.P. Duarte, C. Cavadas, Intracellular signaling mechanisms mediating catecholamine release upon activation of NPY Y1 receptors in mouse chromaffin cells, J. Neurochem. 103 (2007) 896–903. [21] J. Rosmaninho-Salgado, I.M. Araujo, A.R. Alvaro, A.F. Mendes, L. Ferreira,
5. Conclusion Here we demonstrated that inhibition of ERK1/2 in differentiated PC12 cells by U0126 finalized with increased of DA release, decreased the activity of TH and synapsin I, and attenuated expression of SNAP25. Our data also revealed that U0126 treatments led to increasing of PKG activity. Taken together our data confirmed inhibitory role of ERK1/2 and synapsin I in the regulation of DA release from differentiated PC12 cells and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact in the regulation of DA secretion from mature cells. 11
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[25] G.M. Thomas, R.L. Huganir, MAPK cascade signalling and synaptic plasticity, Nat. Rev. Neurosci. 5 (2004) 173–183. [26] R.H. Westerink, A.G. Ewing, The PC12 cell as model for neurosecretion, Acta Physiol. 192 (2008) 273–285. [27] I.A. Yamboliev, L.M. Smyth, L. Durnin, Y. Dai, V.N. Mutafova-Yambolieva, Storage and secretion of beta-NAD, ATP and dopamine in NGF-differentiated rat pheochromocytoma PC12 cells, Eur. J. Neurosci. 30 (2009) 756–768. [28] J. Zhu, T.D. Hexum, Characterization of cocaine-sensitive dopamine uptake in PC12 cells, Neurochem. Int. 21 (1992) 521–526.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
ERK1/2 inhibition increases dopamine release from differentiated PC12 cells D.V. Zosen1, N.A. Dorofeeva, E.V. Chernigovskaya, V.T. Bachteeva, M.V. Glazova
T
⁎
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez pr., 194223 St. Petersburg, Russia
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: PC12 cells Dopamine ERK1/2 U0126 PKG PKA Synapsin I SNAP-25
The release of dopamine (DA) is one of the main steps in the control of neuronal functioning and all CNS. It was demonstrated that many factors such as protein kinases and synaptic proteins are tightly involved in the regulation of DA secretion, but the data are contradictory. Here we analysed an effect of ERK1/2 inhibition on DA secretion from differentiated PC12 cells and evaluated the correlation between the activity of kinases/synaptic proteins and the level of released DA. PC12 cells were differentiated by NGF for 6 days. On the 7th day the cells were incubated for 1, 2 and 4 h with 10μM U0126. Obtained data demonstrated a significant accumulation of DA in the media after 4 h incubation with U0126 that accompanied with upregulation of PKG activity. Analysis of exocytosis proteins demonstrated decreased phosphorylation level of synapsin I and content of SNAP25. Taken together our data proposed an inhibitory role of ERK1/2 in the regulation of catecholamine secretion and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from the cells.
⁎
Corresponding author. E-mail address: [email protected] (M.V. Glazova). Present address: Biocenter Oulu, Laboratory of Developmental Biology, Department of Medical Biochemistry and Molecular Biology, University of Oulu, P.O. Box 5000, Aapistie 5, FIN-90014, Oulu, Finland. 1
https://doi.org/10.1016/j.neulet.2018.06.056 Received 14 March 2018; Received in revised form 25 June 2018; Accepted 28 June 2018 Available online 30 June 2018 0304-3940/ © 2018 Elsevier B.V. All rights reserved.
Neuroscience Letters 684 (2018) 6–12
D.V. Zosen et al.
1. Introduction
2.3. Western blot analysis
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) participate in the regulation of many cellular functions that include proliferation, differentiation, protein synthesis, gene transcription etc. Among ERK1/ 2 substrates tyrosine hydroxylase (TH) is the main rate-limiting enzyme of catecholamine synthesis. ERK1/2 phosphorylates TH at Ser31 and increases enzymatic activity of TH up to two fold. Such a not very impressive increase in enzymatic activity is indeed critical for the regulation of TH basal activity [24]. However, the main regulator of TH activity is cAMP-dependent protein kinase (PKA) that phosphorylates it at Ser40 and activates it up to 15th fold. Other kinases such as Calcium/ calmodulin-stimulated protein kinase II (CaMKII), cyclin-dependent kinase 5 (Cdk5) and cGMP-dependent protein kinase (PKG) also regulate TH activity by phosphorylation at different sites [24]. All these events lead to upregulation of dopamine (DA) synthesis and its accumulation in the neurons that should finalize with DA secretion at the synaptic clefts. However, the mechanisms that regulate catecholamine release are not clearly understood. Many factors could affect on DA release, among these the first discovered and more detailed described are depolarization and intracellular Ca2+ concentration [8]. Study of neurochemical events demonstrated a stimulatory effect of cAMP on catecholamine release [19] and association of PKG activation with upregulation of DA extracellular content [15]. But it is even less known about the participation of ERK1/2 in the regulation of catecholamine release. However, the role of ERK1/2 in the regulation of exocytosis machinery is extensively studied. It was demonstrated that ERK1/2 phosphorylates some cytoskeletal proteins thus participating in exocytosis and synaptic plasticity [25]. To analyze a role of ERK1/2 in DA release we used differentiated PC12 cells. These cells have strong similarity in the synaptic machinery with neurons in the peripheral and central nervous systems that positioned PC12 cells as a perfect model for studding of neurosecretion [26,27]. ERK1/2 is activated by dual-specificity MAP/ERK kinases 1and 2 (MEK1/2), and is the only known substrate for MEK1/2 [22]. Our data demonstrated that treatment with a selective inhibitor of MEK1/2 U0126, which subsequently inhibits ERK1/2, potentiated secretion of DA from cells. Analysis of neurochemical events in the cells showed that inhibition of ERK1/2 was associated with an increase in the activity of cGMP-dependent protein kinase (PKG), but the levels of PKA and CaMKII kinase activity were not affected. Obtained data supposed that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from differentiated mature neurons.
The cells were homogenized in SDS-stop buffer containing 3% betamercaptoethanol and denaturated at 95 °C for 5 min, and when separated on 10% Acrylamide/Bis-Acrylamide gel. The proteins on the gels were transferred to nitrocellulose membranes, which were then incubated overnight with antibody against: TH (#T1299, Sigma), p-TH (Ser31, #AB5423, Millipore), ERK1/2 (#9102, Cell Signaling), pERK1/2 (#4376, Cell Signaling), synapsin I (#AB1543 P, Millipore), psynapsin I (Ser 62/67, #AB9848, Millipore), SNAP25 (#MAB331, Chemicon), VAMP-2 (#104221, Synaptic System), p-CaMKII (Thr286, #MA 1-047, Thermo Scientific), p-VASP Ser239 (#sc-101439, Santa Cruze Biotech.) or p-VASP Ser157 (#sc-101440, Santa Cruze Biotech.) followed by incubation with anti-rabbit or anti-mouse HRP secondary antibody and final chemilumeniscent detection by Super Signal West Dura Extended Duration Substrate (#34075, Thermo Scientific). 2.4. Statistics Statistical analysis was carried out by the Student’s t-test, and values are expressed as mean ± SE for ELISA, and for western blot analysis. p < 0.05 was taken as levels of statistical significance. 3. Results 3.1. Analysis of DA concentration Firstly, we determined DA concentration in the media after 1, 2 and 4 h’ incubation with U0126. Obtained results demonstrated the increasing level of DA in 4 h’ incubation of differentiated PC12 cells with U0126 (Fig. 1) that proposed upregulation of DA release from the cells. 3.2. Effects of U0126 on ERK1/2 and TH phosphorylation After application of U0126 phosphorylation of ERK1/2 was completely disappeared (Fig. 2A). Then we analyzed phosphorylation level of TH at Ser31, which is specific site mainly for ERK1/2 [11]. The results showed significantly decreased phosphorylation of TH at Ser31 at all-time points (Fig. 2B) that revealed catecholamine down-regulated basal secretion. We also obtained an interesting result that in control groups ERK1/2 phosphorylation levels were upregulated in a time-dependent manner (Fig. 2A) and the same changes ware estimated for TH phosphorylation (Fig. 2B), supposing some compensatory mechanism on short-term NGF omission.
2. Material and methods 2.1. Cell culture PC12 cells were incubated on collagen-coated plates (collagen rat tail #11 179 179 001, Roche) in DMEM supplemented with 10% horse serum, 5% FBS, penicillin- streptomycin (all from Sigma). When the cell confluence reached about 50–60% the medium was changed to DMEM supplemented with 1% horse serum and NGF (50 ng/ml, #01-125, Millipore) for stimulation of differentiation. The cells were incubated with NGF for 6 days. On the next day the medium was changed for DMEM only and 10 μM U0126 (#1144, Tocris) was added for 1, 2 and 4 h (n = 6 for each time point). At each time point, media was collected for further ELISA analysis, the cells were homogenized for Western blotting. 2.2. ELISA
Fig. 1. ERK1/2 inhibition stimulates DA release from differentiated PC12 cells. DA concentration (ng/ml) in media after incubation the cells with U0126 during 1, 2 and 4 h. Data are shown as means ± SE. * p < 0.05 versus corresponding control.
DA concentrations in the media were determined by DA ELISA kit (#40-371-25013, GenWay Biothech Inc., San Diego, CA, USA) according to the manufacturer instruction. 7
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Fig. 2. Analysis the phosphorylation levels of ERK1/2 and TH after U0126 treatments. PC12 cells were incubated for 1, 2 and 4 h with U0126 and phosphorylation of ERK1/2 (A) and TH at Ser31 (B) were estimated by Western blot. Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. * p < 0.05 versus corresponding control.
phosphorylation levels was detected in both control and U0126 treated groups (Fig. 3B) in comparison with the groups after 1 h incubation. On the other hand, phosphorylation level of VASP at Ser239 was increased after application of U0126 (Fig. 3C) that proposed upregulation of PKG activity.
3.3. Activity of PKA, PKG and CaMKII We analyzed of PKA, PKG and CaMKII activity by estimation of its phosphorylation levels. Obtained results of CaMKII phosphorylation level at Thr286, which corresponds to CaMKII activation, demonstrated that inhibition of ERK1/2 did not affect it (Fig. 3A). The activity of PKA and PKG was analyzed by estimation of phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) at Ser157 and Ser239, which are specific phosphorylation sites for PKA and PKG correspondently [3]. Expression of VASP protein was demonstrated in many cell types including neurons and PC12 cells where this protein plays a significant role in the regulation the neurite growth cone formation [10]. Our results demonstrated that phosphorylation level of VASP at Ser157 was not affected by U0126 treatment (Fig. 3B) displaying unchanged activity of PKA in comparison with corresponding control groups. However, after 2 and 4 h, significant reduction of VASP Ser157
3.4. Analysis of exocytosis proteins We studied synapsin I protein expression and phosphorylation levels of synapsin I at Ser 62/67, which, like TH, are also the targets for ERK1/2 [4]. While pan synapsin I level was not altered by decreased activity of ERK1/2, the level of synapsin I phosphorylation was significantly decreased (Fig. 4B). Then we analyzed synaptosomal-associated protein 25 (SNAP-25) and vesicle-associated membrane protein 2 (VAMP2) proteins, which belong to SNARE complex. Obtained data demonstrated unchanged protein level of VAMP2 in the cells (data not 8
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Fig. 3. Analysis of the activity of CaMKII, PKA and PKC kinases in differentiated PC12 cells after inhibition of ERK1/2 activity. A – phosphorylation of CaMKII was not changed in the cells after inhibition of ERK1/2 activity. B, C – analysis of PKA and PKC activity after inhibition of ERK1/2 were estimated by phosphorylation levels of VASP at Ser157 (phosphorylation site for PKA) (B), and at Ser 239 (phosphorylation site for PKG) (C). Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. * p < 0.05; ** p < 0.01 versus corresponding control. 9
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Fig. 4. U0126 treatment significantly decreases SNAP-25 expression and phosphorylation of synapsin I. A – expression SNAP-25 protein in the differentiated PC12 cells after incubation with U0126 during 1, 2 and 4 h. B – phosphorylation level of synapsin I at Ser62/67 estimated by Western Blot. Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. * p < 0.05; ** p < 0.01 versus corresponding control.
neurons after inhibition of ERK1/2 by U0126. Thus the authors proposed the inhibitory role of ERK1/2 in the regulation of neurotransmitter release. These data are in line with our observation and confirmed that ERK1/2 repress unstimulated basal secretion of DA in differentiated PC12 cells. We also could not exclude the possibility that accumulation of DA in the media may be caused by inhibition of DA reuptake. Published data demonstrated that an inhibition of ERK1/2 prevents DA uptake in in vitro experiments on isolated synaptosomes [17]. However, Lin with co-authors did not show any effects of U0126 on DA re-uptake in DAT transfected cells [16]. It is known that cocaine blocks DA re-uptake in PC12 cells [1,28], but on the other hand cocaine also significantly increases activity of ERK1/2 [12,14]. So it seems that ERK1/2-dependent regulation of DA re-uptake could be dependent from the cell types or cell environment since in synaptosomes ERK1/2 blockade inhibits DA re-uptake, while cocaine, which activates ERK1/2,
shown). However, the level of SNAP-25 protein was significantly decreased (Fig. 4A). 4. Discussion Here we analyzed a role of ERK1/2 kinase in the regulation of DA release from differentiated PC12 cells. Analysis of DA concentration in the media after inhibition of ERK1/2 demonstrated the increasing level of DA in 4 h’ incubation of differentiated PC12 cells that proposed upregulation of DA release from the cells. In spite of plenty published data [2,5,6,20,21] demonstrating a positive effect of ERK1/2 in the regulation of induced catecholamine secretion our data revealed that inhibition of ERK1/2 activity potentiated the release of DA from differentiated PC12 cells. Recently Subramanian and Morozov [23] also revealed a stimulation of vesicular exocytosis in primary hippocampal 10
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Conflict of interest
has got an inhibitory effect on DA re-uptake too. Summarizing published data, we could not exclude effect of ERK1/2 inhibition on DA reuptake in differentiated PC12 cells. According the arising complexity of these process and difficulties to predict the effect, this question should be addressed to further studies. Then we analyzed ERK-dependent activity of TH, which is the main rate-limiting enzyme of catecholamine synthesis. After U0126 treatments the phosphorylation level of TH at Ser31 was decreased. Such significant elimination of TH phosphorylation at Ser31 could be dependent on ERK1/2 inactivation [24]. However, we did not see complete dephosphorylation of TH as we observed for ERK1/2 that can be explained by the fact of TH phosphorylation at Ser31 also by Cdk5 [24]. We also found an interesting fact that in control groups ERK1/2 phosphorylation levels were upregulated in a time-dependent manner and the same changes were estimated for TH phosphorylation, supposing some compensatory mechanism on short-term NGF omission. Moreover, our data demonstrated that decreased ERK-dependent activity of TH and respectively decreased basal synthesis of DA did not affect the fast release of DA from the cells. It is known that exocytosis of neurotransmitters could be dependent on the activity of PKA, PKG and CaMKII protein kinases. Obtained results demonstrated that inhibition of ERK1/2 did not affect on CaMKII activity level as well as on the activity of PKA. However, our data revealed activation of PKG. PKG highly expresses in the brain and plays a significant role in the regulation of neurotransmitter release, in particular, DA secretion [24]. It would also need to mention that in control group after 4 h’ incubation an increased activity of PKG correlated with the enhanced level of DA in comparison with the 1-hour control group after. Exocytosis machinery is operated by many exocytosis related proteins such as synapsins and soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins. We analyzed if the inhibition of ERK1/2 correlated with some alteration in the exocytosis proteins therefor promoting DA release. Initially we studied synapsin I protein expression and phosphorylation levels of synapsin I at Ser 62/ 67, which, like TH, are also the targets for ERK1/2 [4]. While pan synapsin I level was not altered by decreased activity of ERK1/2, the level of synapsin I phosphorylation was significantly decreased. It is known that synapsins regulate neurotransmitter release. However, its role could be variable. ERK1/2-dependent synapsin I phosphorylation positively regulates glutamate release [4], while Kile with co-authors demonstrated that synapsins play an inhibitory role in DA secretion in vivo and in vitro [13]. Our data confirmed this observation since ERK1/2 dependent inhibition of synapsin I correlated with increased DA secretion. Then we analyzed synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) proteins, which belong to SNARE complex. Obtained data demonstrated unchanged protein level of VAMP2 in the cells (data not shown). However, the level of SNAP-25 protein was significantly decreased. These data corresponds to our previous data, which demonstrated a correlation of SNAP-25 protein level with the level of vasopressin [18] and glutamate release [9]. Moreover, Eisenhofer and co-authors also revealed the same correlation between catecholamine release and expression of SNAP-25 [7].
The authors declare that they have no conflict of interest. Acknowledgments This work was supported by FASO of Russia (#АААА-А18118012290371-3). Part of the analysis was done at Research Resource Center #441590 at Sechenov Institute of Evolutionary Physiology and Biochemistry. References [1] R.B. Badisa, C.S. Batton, E. Mazzio, S.C. Grant, C.B. Goodman, Identification of biochemical and cytotoxic markers in cocaine treated PC12 cells, Sci. Rep. 8 (2018) 2710. [2] E. Bloch-Shilderman, H. Jiang, S. Abu-Raya, M. Linial, P. Lazarovici, Involvement of extracellular signal-regulated kinase (ERK) in pardaxin-induced dopamine release from PC12 cells, J. Pharmacol. Exp. Ther. 296 (2001) 704–711. [3] E. Butt, K. Abel, M. Krieger, D. Palm, V. Hoppe, J. Hoppe, U. Walter, cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets, J. Biol. Chem. 269 (1994) 14509–14517. [4] F. Cesca, P. 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5. Conclusion Here we demonstrated that inhibition of ERK1/2 in differentiated PC12 cells by U0126 finalized with increased of DA release, decreased the activity of TH and synapsin I, and attenuated expression of SNAP25. Our data also revealed that U0126 treatments led to increasing of PKG activity. Taken together our data confirmed inhibitory role of ERK1/2 and synapsin I in the regulation of DA release from differentiated PC12 cells and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact in the regulation of DA secretion from mature cells. 11
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