Differential Modulation of Akt/Glycogen Synthase Kinase-3β Pathway Regulates Apoptotic and Cytoprotective Signaling Responses
2008; Elsevier BV; Volume: 283; Issue: 22 Linguagem: Inglês
10.1074/jbc.m707238200
ISSN1083-351X
AutoresVenugopalan D. Nair, C. Warren Olanow,
Tópico(s)Cancer-related gene regulation
ResumoWe have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the pro-apoptotic factor glycogen synthase kinase-3β (GSK-3β). Ropinirole-induced protection was associated with phosphorylation of GSK-3β (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3β (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3β and reduced levels of β-catenin, a transcriptional activator that is regulated by GSK-3β. Knock-out of GSK-3β expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H2O2) dephosphorylates Akt and GSK-3β, increases GSK-3β activity, and promotes an interaction with β-catenin and its degradation. Inhibition of GSK-3β activity by inhibitor VIII protects cells from H2O2 similar to ropinirole. These results indicate that GSK-3β downstream of Akt plays a critical role in cell death and survival in these models. We have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the pro-apoptotic factor glycogen synthase kinase-3β (GSK-3β). Ropinirole-induced protection was associated with phosphorylation of GSK-3β (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3β (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3β and reduced levels of β-catenin, a transcriptional activator that is regulated by GSK-3β. Knock-out of GSK-3β expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H2O2) dephosphorylates Akt and GSK-3β, increases GSK-3β activity, and promotes an interaction with β-catenin and its degradation. Inhibition of GSK-3β activity by inhibitor VIII protects cells from H2O2 similar to ropinirole. These results indicate that GSK-3β downstream of Akt plays a critical role in cell death and survival in these models. The characteristic pathology of Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; PI-3K, phosphoinositide 3-kinase; Akt, protein kinase B; PH, pleckstrin homology; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; GSK-3β, glycogen synthase kinase-3β; shRNA, short hairpin RNA; FKHR, fork head transcription factor; 6-OHDA, 6-hydroxydopamine; ERK, extracellular-regulated kinase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein; GST, glutathione S-transferase; D2R, D2 receptor. is degeneration of dopaminergic neurons coupled with Lewy body inclusions in the substantia nigra pars compacta (1Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1256) Google Scholar). The mechanism underlying dopaminergic cell death in PD has not been elucidated. A variety of cellular and molecular changes indicative of mitochondrial dysfunction, oxidative stress, proteasomal dysfunction, and apoptosis have been identified in the parkinsonian brain (for review, see Refs. 2Moore D.J. West A.B. Dawson V.L. Dawson T.M. Annu. Rev. Neurosci. 2005; 28: 57-87Crossref PubMed Scopus (1053) Google Scholar and 3Olanow C.W. Annu. Rev. Med. 2004; 55: 41-60Crossref PubMed Scopus (159) Google Scholar). Specifically, a large body of evidence suggests that oxidative stress or reactive oxygen species-mediated apoptosis may contribute to the progressive and selective neuronal degeneration observed in PD (4Beal M.F. Ann. N. Y. Acad. Sci. 2003; 991: 120-131Crossref PubMed Scopus (469) Google Scholar). Brains of PD patients have increased iron, which promotes free radical formation, decreased levels of reduced glutathione, which is the major anti-oxidant in the brain, and evidence of oxidative damage to DNA, lipids, and proteins (5Teismann P. Vila M. Choi D.K. Tieu K. Wu D.C. Jackson-Lewis V. Przedborski S. Ann. N. Y. Acad. Sci. 2003; 991: 272-277Crossref PubMed Scopus (161) Google Scholar). Furthermore, in the substantia nigra pars compacta of PD patients there are increased levels of cyclooxygenase, which contribute to formation of the oxidant species dopamine-quinone (5Teismann P. Vila M. Choi D.K. Tieu K. Wu D.C. Jackson-Lewis V. Przedborski S. Ann. N. Y. Acad. Sci. 2003; 991: 272-277Crossref PubMed Scopus (161) Google Scholar), and reduced mitochondrial complex I activity, which promotes free radical formation (6Schapira A. Cooper J. Dexter D. Clark J. Jenner P. Marsden C. J. Neurochem. 1990; 54: 823-827Crossref PubMed Scopus (1681) Google Scholar, 7Parker Jr., W.D. Boyson S.J. Parks J.K. Ann. Neurol. 1989; 26: 719-723Crossref PubMed Scopus (964) Google Scholar, 8Keeney P.M. Xie J. Capaldi R.A. Bennett Jr., J.P. J. Neurosci. 2006; 26: 5256-5264Crossref PubMed Scopus (588) Google Scholar). Current therapies for PD are primarily based on a dopamine replacement strategy. Although they provide effective anti-parkinsonian effects, particularly in the early stages of the disease, PD patients eventually develop potentially disabling features such as falling, freezing, and dementia that are not satisfactorily controlled with available therapies (9Olanow C.W. Obeso J.A. Stocchi F. Lancet Neurol. 2006; 5: 677-687Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). As a consequence, there has been an intensive search for therapies that might protect or restore function to neurons that would otherwise undergo degeneration in PD and thereby stop or slow the rate of disease progression. Dopamine agonists that activate D2 receptors are widely used to treat PD based on their capacity to provide short-term symptomatic improvements. Recent interest has also focused on the potential of dopamine agonists to provide neuroprotective effects and slow the rate of PD progression (18Olanow C.W. Jenner P. Brooks D. Ann. Neurol. 1998; 44: 167-174Crossref PubMed Scopus (149) Google Scholar). Ropinirole and other dopamine agonists have been found to be capable of protecting dopamine neurons from a variety of toxins in both in vitro and in vivo models (10Du F. Li R. Huang Y. Li X. Le W. Eur. J. Neurosci. 2005; 22: 2422-2430Crossref PubMed Scopus (107) Google Scholar, 11Joyce J.N. Presgraves S. Renish L. Borwege S. Osredkar T. Hagner D. Replogle M. PazSoldan M. Millan M.J. Exp. Neurol. 2003; 184: 393-407Crossref PubMed Scopus (86) Google Scholar, 12Millan M.J. Di Cara B. Hill M. Jackson M. Joyce J.N. Brotchie J. McGuire S. Crossman A. Smith L. Jenner P. Gobert A. Peglion J.L. Brocco M. J. Pharmacol. Exp. Ther. 2004; 309: 921-935Crossref PubMed Scopus (44) Google Scholar, 13Presgraves S.P. Borwege S. Millan M.J. Joyce J.N. Exp. Neurol. 2004; 190: 157-170Crossref PubMed Scopus (53) Google Scholar, 14Nair V.D. Olanow W. Sealfon S.C. Biochem. J. 2003; 373: 25-32Crossref PubMed Scopus (73) Google Scholar, 15Nair V.D. Sealfon S.C. J. Biol. Chem. 2003; 278: 47053-47061Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Furthermore, in clinical trials in PD patients, ropinirole delayed the rate of decline of a neuroimaging surrogate biomarker of nigrostriatal function in comparison to levodopa (16Whone A.L. Watts R.L. Stoessl A.J. Davis M. Reske S. Nahmias C. Lang A.E. Rascol O. Ribeiro M.J. Remy P. Poewe W.H. Hauser R.A. Brooks D.J. Ann. Neurol. 2003; 54: 93-101Crossref PubMed Scopus (789) Google Scholar, 17Brunt E.R. Brooks D.J. Korczyn A.D. Montastruc J.L. Stocchi F. J. Neural Transm. 2002; 109: 489-502Crossref PubMed Scopus (59) Google Scholar). These findings raise the possibility that ropinirole may be neuroprotective and slow the rate of PD progression. Although several mechanisms have been proposed to account for how these agents might provide neuroprotection (18Olanow C.W. Jenner P. Brooks D. Ann. Neurol. 1998; 44: 167-174Crossref PubMed Scopus (149) Google Scholar), most interest has focused on the potential of their capacity to provide anti-apoptotic effects. However, the precise signaling mechanism whereby ropinirole induces anti-apoptotic effects is not known. Previously, we have demonstrated that some dopamine agonists protect PC12 cells from oxidative stress by activating a D2 receptor-dependent PI-3K/Akt signaling pathway independent of their ability to activate GTPγS binding (14Nair V.D. Olanow W. Sealfon S.C. Biochem. J. 2003; 373: 25-32Crossref PubMed Scopus (73) Google Scholar, 15Nair V.D. Sealfon S.C. J. Biol. Chem. 2003; 278: 47053-47061Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To elucidate the downstream effectors of the PI-3K/Akt signaling pathway that mediates the agonist-specific modulation of cell survival, we have investigated the anti-apoptotic signaling pathway activated by the dopamine agonist ropinirole. We now report that ropinirole-mediated protection against oxidative stress involves activation of PI-3K/Akt-mediated phosphorylation (deactivation) of GSK-3β and that oxidative stress has opposing effects on the modulation of this pathway. Chemicals—Dulbecco'ns modified Eagle'ns medium (DMEM), Opti-MEM1, DMEM/F-12, N2 supplements, and fetal calf serum were obtained from Invitrogen. Ropinirole was obtained from GlaxoSmithKline Inc (Research Triangle Park, NC). PI-3K inhibitor LY294002 and GSK-3β inhibitor VIII were from Calbiochem. CellTiter-Blue assay kits were from Promega (Madison, WI). An Amersham Biosciences enhanced chemiluminescence lighting (ECL) Western blotting detection reagent kit was from GE Healthcare. Haloperidol, eticlopride, hydrogen peroxide (H2O2), poly-d-lysine, and β-actin antibody were from Sigma. Antibodies specific to phospho-Akt, Akt, phospho-GSK-3β, phospho-p70S6 kinase, phospho-β-catenin, and β-catenin were from Cell Signaling Technology (Beverly, MA). Phospho-Forkhead transcription factor (FKHR), and GSK-3β antibodies were from Santa Cruz Biotechnology (Santa Clara, CA). Tyrosine hydroxylase antibody was from Chemicon (Temecula, CA). Neural Transit transfection reagent was from Mirus (Madison, WI). Alexa Fluor 488 donkey anti-sheep IgG and Alexa Fluor 592 chicken anti-rabbit IgG were from Molecular Probes (Invitrogen). [3H]Dopamine (40 μCi/mmol) was purchased from Amersham Biosciences. Cell Cultures—PC12 and PC12-D2R cells were cultured as previously described in a humidified atmosphere containing 5% CO2 at 37 °C (14Nair V.D. Olanow W. Sealfon S.C. Biochem. J. 2003; 373: 25-32Crossref PubMed Scopus (73) Google Scholar, 15Nair V.D. Sealfon S.C. J. Biol. Chem. 2003; 278: 47053-47061Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19Nair V.D. Apoptosis. 2006; 11: 955-966Crossref PubMed Scopus (72) Google Scholar, 20Nair V.D. McNaught K.S. Gonzalez-Maeso J. Sealfon S.C. Olanow C.W. J. Biol. Chem. 2006; 281: 39550-39560Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 21Nair V.D. Yuen T. Olanow C.W. Sealfon S.C. J. Biol. Chem. 2004; 279: 27494-27501Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Medium was replaced with Opti-MEM1 3 h before various treatments of the cells. Dopaminergic neuronal cultures were prepared from embryonic day 14 rat fetuses (E14; Charles River Laboratories, Wilmington, MA) as previously described (22Bouvier M.M. Mytilineou C. J. Neurosci. 1995; 75: 7141-7149Crossref Google Scholar). Briefly, the ventral portion of the midbrain was removed in sterile ice-cold Ca2+ and Mg2+-free HBSS/HEPES solution, cleaned free of meningeal tissue, and mechanically dissociated by passage through a flame-polished Pasteur pipette. Dissociated cells were plated at a density of ∼1.2 × 105 cells per cm2 on poly-d-lysine-coated 24-well plates or glass coverslips. The neurons were maintained in a chemically defined medium consisting of Dulbecco'ns modified Eagle'ns medium/F-12 medium with N2 supplements, l-glutamine (0.5 mm), and penicillin/streptomycin (serum-free medium). Half of the culture medium was replaced every 2 days. Approximately 5-7-day-old cultures were used for experiments. CellTiter-Blue Cytotoxicity Assays—For analysis of cell survival, cells were plated at a density of 104 cells/well on 96-microwell cell culture plates (in 100 μl of medium) and grown for 24 h. Thereafter, 200 μm H2O2 was added either with or without ropinirole at the indicated concentrations, and cells were incubated for another 24 h. After treatments, 20 μl of the Cell-Titer-Blue reagent was added to each well, and the plates were incubated for 2-3 h. The conversion of non-fluorescent Cell-Titer-Blue reagent to highly fluorescent substrate by living cells was quantified using a spectrofluorometer (Spectra Max Gemini XS, Molecular Devices, Sunnyvale, CA) as described (19Nair V.D. Apoptosis. 2006; 11: 955-966Crossref PubMed Scopus (72) Google Scholar, 20Nair V.D. McNaught K.S. Gonzalez-Maeso J. Sealfon S.C. Olanow C.W. J. Biol. Chem. 2006; 281: 39550-39560Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The data are expressed as percentages of the vehicle-treated controls, and the values represent the means ± S.E. from eight microwells from each of three independent experiments (n = 24). Uptake of [3H]Dopamine—[3H]Dopamine uptake by primary mesencephalic neurons was carried out as described previously (19Nair V.D. Apoptosis. 2006; 11: 955-966Crossref PubMed Scopus (72) Google Scholar). The results were expressed as percentages of vehicle-treated control culture response. Transfections and DNA Constructs—PC12-D2R cells (1 × 105) were plated into 60-mm culture dishes with complete medium. After 24 h, the cells were transfected with 2.5 μg of the plasmid DNA encoding the pleckstrin homology (PH) domain of Akt protein kinase (1-167 amino acids) tagged with green fluorescent protein (PH-Akt-GFP) (15Nair V.D. Sealfon S.C. J. Biol. Chem. 2003; 278: 47053-47061Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) or shRNA constructs such as U6-XASH3 HP, U6-Akt1 HP3, and U6-GSK-3β HP1 (generously provided by D. L. Turner (23Yu J.Y. Taylor J. DeRuiter S.L. Vojtek A.B. Turner D.L. Mol. Ther. 2003; 7: 228-236Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar)) with the use of the TransIT-Neural transfection reagent as described previously (19Nair V.D. Apoptosis. 2006; 11: 955-966Crossref PubMed Scopus (72) Google Scholar). Immunocytochemistry—For immunofluorescence detection, PC12-D2R cells expressing PH-Akt-GFP cells were treated with ropinirole for 15 min. The cells were fixed, and GFP was visualized under an Olympus (BX65) upright epifluorescence microscope. To detect endogenous phospho-Akt, phospho-GSK-3β, GSK-3β, and β-catenin, PC12-D2R cells were immunostained with antibodies to phospho-Akt, phospho-GSK-3β, GSK-3β, or β-catenin and visualized using Alexa Fluor 592-conjugated chicken anti-rabbit IgG as described previously (20Nair V.D. McNaught K.S. Gonzalez-Maeso J. Sealfon S.C. Olanow C.W. J. Biol. Chem. 2006; 281: 39550-39560Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To detect phospho-GSK-3β in dopamine neurons, primary mesencephalic cultures were stained with antibodies specific to tyrosine hydroxylase and phospho-GSK-3β and visualized using Alexa Fluor 488 donkey anti-sheep IgG and 592-conjugated chicken anti-rabbit IgG. The nuclei were stained with fluorescent DNA dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) mounting medium and examined under an Olympus (BX65) upright epifluorescence microscope. Immunoblotting and Immunoprecipitation—Cells (3 × 106 cells/100-mm plate) were grown for 24 h, and after respective treatments, the cells were washed twice with ice-cold phosphate-buffered saline and lysed in buffer 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% IGEPAL C630, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 5 μg/ml aprotinin, and a mixture of protease inhibitors (Roche Diagnostics) at 4 °C for 20 min. After centrifugation at 14,000 × g for 20 min at 4 °C, equal amounts of proteins were resolved by SDS-polyacrylamide gel electrophoresis. The resolved proteins were electrotransferred to nitrocellulose membrane and incubated with phosphorylated Akt, Akt substrates, GSK-3β, p70S6 kinase, FKHR, or β-catenin and then detected with peroxidase-conjugated secondary antibodies and ECL reagent. The blots were then stripped in stripping buffer containing 62.5 mm Tris-HCl, pH 6.7, 2% SDS, 100 μm β-mercaptoethanol, and probed for total Akt, GSK-3β, or β-actin proteins. For immunoprecipitation, the protein extract was incubated sequentially (2 h for each incubation at 4 °C) with anti-GSK-3β antibody and protein A/G-agarose (Santa Cruz Biotechnology) with gentle agitation. Immunoprecipitates were washed 3 times with lysis buffer, boiled for 5 min in 3× Laemmli sample buffer, and processed for Western blotting using GSK-3β or β-catenin antibody. The blots were stripped and reprobed with anti-β-actin antibodies. In Vitro GSK-3β Activity Assay—GSK-3β was immunoprecipitated at 4 °C from 1 mg of protein extract with 2 μg of anti-GSK-3β antibody and anti-rabbit IgG beads (eBioscience, San Diego, CA). Bacterial lysate for GST-β-catenin fusion protein (24Wei Y. Renard C.A. Labalette C. Wu Y. Levy L. Neuveut C. Prieur X. Flajolet M. Prigent S. Buendia M.A. J. Biol. Chem. 2003; 278: 5188-5194Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) was pre-bound to GST-Sepharose beads (Amersham Biosciences) at room temperature. Beads were then incubated with GSK-3β immunoprecipitate in assay buffer (20 mm Tris, pH 7.5, 10 mm MgCl2, 5 mm dithiothreitol, 0.2 mm ATP) for 30 min at 30 °C. Reactions were stopped by the addition of Laemmli loading buffer, boiled for 5 min, and resolved on SDS-PAGE. Western immunoblotting was carried out using Rabbit IgG TrueBlot Set reagents (eBioscience). Anti-β-catenin and anti-phospho-β-catenin antibodies were used to detect GST-β-catenin. To detect GSK-3β in the reaction mixture, anti-GSK-3β antibody was used. Statistical Analysis—Data were analyzed by either two-tailed t test or analysis of variance followed by the Tukey'ns test to correct for multiple comparisons. The Dopamine Agonist Ropinirole Protects PC12-D2R Cells and Primary Mesencephalic Neurons from Cell Death Induced by Oxidative Stress via Activation of the D2 Receptors—We studied the potential of the dopamine agonist ropinirole to protect against apoptosis induced by H2O2 in PC12 cells that express D2 receptors (PC12-D2R). Cells were preincubated (1 h) with varying concentrations of ropinirole (10-11 to 10-3 m) before the addition of H2O2 (200 μm). After 24 h of incubation, cell viability was assessed using the CellTiter-Blue cell death assay. The administration of 200 μm of H2O2 induced a 52.9 ± 5.0% reduction in cell survival in comparison to controls. Ropinirole protected PC12-D2R cells from H2O2-induced apoptosis in a robust and concentration-dependent manner (Fig. 1A). In contrast, ropinirole did not protect PC12 cells that lacked D2 receptors from exposure to H2O2. Ropinirole also did not protect against H2O2 when PC12-D2R cells were pretreated with the dopamine antagonist haloperidol (10 μm) before the addition of ropinirole (Fig. 1B). Although PC12 cells are used as a good model to study dopaminergic function, they are non-neuronal cells derived from adrenal pheochromocytomas. Therefore, to further determine whether ropinirole protects dopamine neurons, we used primary rat mesencephalic neuronal cultures treated with 6-hydroxydopamine (6-OHDA). As shown in Fig. 1, C and D, ropinirole (1 μm) offered significant neuroprotection against 6-OHDA-induced neuronal loss. Our results are consistent with the report that ropinirole protects the primary mesencephalic neurons from 1-methyl-4-phenylpyridinium, the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine toxicity (10Du F. Li R. Huang Y. Li X. Le W. Eur. J. Neurosci. 2005; 22: 2422-2430Crossref PubMed Scopus (107) Google Scholar). Ropinirole did not protect against 6-OHDA toxicity when primary mesencephalic neurons were co-incubated with ropinirole plus the dopamine antagonist eticlopride (10 μm) (Fig. 1B). These results indicate that ropinirole protects dopamine neurons against cell death induced by H2O2 and 6-OHDA and that this protection occurs by way of functional D2 receptors. Ropinirole Induced Protection in PC12-D2R Cells, and Primary Mesencephalic Neurons Involves the PI-3K/Akt Signaling Pathway—We have previously reported that the increase in cell survival mediated by D2 receptor activation is abolished by inhibitors of PI-3K (14Nair V.D. Olanow W. Sealfon S.C. Biochem. J. 2003; 373: 25-32Crossref PubMed Scopus (73) Google Scholar). We, therefore, studied whether PI-3K signaling is modulated by the D2 receptor when complexed with ropinirole. To determine whether PI-3K signaling is involved in ropinirole-mediated protection, we tested the effect of the PI-3K inhibitor LY294002 (10 μm). Inhibition of PI-3K completely abolished the capacity of ropinirole to protect against cell death induced by oxidative stress in PC12 cells and by 6-OHDA in primary mesencephalic neuronal cultures (Fig. 1, B-D). However, LY294002 by itself had no effect on cell survival (Fig. 1B). These data suggest that activation of the PI-3K pathway contributes to the protective effects of ropinirole against apoptosis induced by oxidative stress in PC12-D2R cells and by 6-OHDA in primary mesencephalic neuronal cultures. To examine if Akt, the principle downstream target of PI-3K (25Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar), is implicated in ropinirole-mediated neuroprotection, we measured the translocation and phosphorylation of Akt after ropinirole administration. Akt phosphorylation and its protective effects occur after it translocates to the plasma membrane through an interaction of its N-terminal PH domain with phosphatidylinositol 3,4,5-triphosphate (PIP3) (26Datta K. Franke T.F. Chan T.O. Makris A. Yang S.I. Kaplan D.R. Morrison D.K. Golemis E.A. Tsichlis P.N. Mol. Cell. Biol. 1995; 15: 2304-2310Crossref PubMed Scopus (157) Google Scholar), thereby bringing the enzyme into the proximity of additional PIP3-dependent and -independent protein kinases (27Bellacosa A. Chan T.O. Ahmed N.N. Datta K. Malstrom S. Stokoe D. McCormick F. Feng J. Tsichlis P. Oncogene. 1998; 17: 313-325Crossref PubMed Scopus (458) Google Scholar). The distribution of Akt was assessed using a PH-Akt-GFP (15Nair V.D. Sealfon S.C. J. Biol. Chem. 2003; 278: 47053-47061Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In unstimulated cells, phosphorylated Akt was mainly localized in the perikarya (supplemental Fig. 1). In contrast, D2 receptor activation by ropinirole caused a rapid (15 min) translocation of PH-Akt-GFP to peripheral membrane regions (Fig. 2A, right panel). Ropinirole-induced translocation was similarly demonstrated using antibodies to endogenous phospho-Akt (Fig. 2, B and C). The addition of ropinirole to normal PC12-D2R cells was also associated with a significant increase in phosphorylated Akt, with phosphorylation occurring at the serine 473 (Ser473) site. These results were also observed 15 min after the addition of the drug and returned to basal levels at 60 min (Fig. 2, D and E). Levels of total Akt protein remained unchanged. Thus, D2 receptor stimulation by ropinirole in PC12-D2R cells causes rapid translocation and phosphorylation of Akt. These changes in Akt translocation and phosphorylation were prevented by co-administration of the PI-3K inhibitor LY294002 (data not shown), indicating that ropinirole induces Akt activation through a PI-3K signaling pathway. Effects of Ropinirole on Downstream Effectors of PI-3K/Akt Signaling Pathway—The PI-3K/Akt pathway is known to promote cell survival by inactivating pro-apoptotic factors and activating anti-apoptotic factors by phosphorylation-dependent mechanisms (28Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4957) Google Scholar, 29del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1989) Google Scholar, 30Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). To determine whether ropinirole induces the activation of any specific effectors of the PI-3K/Akt pathway, we examined the effect of ropinirole on the phosphorylation at Ser/Thr of Akt substrate proteins (28Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4957) Google Scholar) when administered to untreated PC12-D2R cells. Western blot analysis demonstrated that ropinirole treatment induced a transient accumulation of phosphorylated Akt substrates coincident with the timing of the phosphorylation of Akt (Fig. 3A). One of the phosphorylated downstream substrates of Akt was established to be GSK-3β (Fig. 3, B and C), which in its activated (dephosphorylated) state promotes cell death in response to oxidative stress (31Castelo-Branco G. Rawal N. Arenas E. J. Cell Sci. 2004; 117: 5731-5737Crossref PubMed Scopus (127) Google Scholar). Ropinirole did not induce phosphorylation of p70S6 kinase or FKHR (Fig. 3B), two other downstream substrates of Akt. Blockade of PI-3K by LY294002 blocked the ropinirole-induced phosphorylation of Akt substrates including GSK-3β (Fig. 4).FIGURE 4Inhibition of PI 3-kinase abolished the phosphorylation of Akt and effectors of Akt by ropinirole. PC12-D2R cells were pretreated with PI-3K inhibitor LY294002 (10 μm) for 30 min and incubated with 1 μm of ropinirole for 15 min. After stimulation, cells lysates were prepared and analyzed by Western immunoblotting with specific antibodies anti-phospho (p)-Akt, phospho-(Ser/Thr) Akt substrates, and phospho-GSK-3β (Ser9) antibodies. Phospho-GSK-3β blot was stripped and reprobed with anti-β-actin antibody. Experiments were repeated three times with similar results. Note that ropinirole induces phosphorylation of Akt and GSK-3β and that these effects are reversed with the PI-3K inhibitor LY294001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Akt and GSK-3β Are Essential for Cell Survival—To investigate the role of Akt/GSK-3β signaling in cell survival, shRNA specific to Akt1 and GSK-3β were used (23Yu J.Y. Taylor J. DeRuiter S.L. Vojtek A.B. Turner D.L. Mol. Ther. 2003; 7: 228-236Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We used a PH-Akt-GFP construct to determine the transfection efficiency of PC12-D2R cells. Transient expression of PH-Akt-GFP yielded ∼50-60% GFP-positive cells after 48 h of transfection (data not shown). When normal PC12-D2R cells were transfected with either Akt shRNA or GSK-3β shRNA, markedly reduced cell viability was observed 48 h after transfection (Fig. 5, A and B). When compared with transfection of the U6-XASH3 HP control vector, transfection of hairpin small interfering RNA expression vectors against Akt and GSK-3β reduced the expression of Akt and GSK-3β, respectively (Fig. 5, A and B). Because GSK-3β is a downstream effector of Akt, we examined the phosphorylation of GSK-3β in Akt knockdown cells. As shown in Fig. 5C, Akt suppression reduced phosphorylation of GSK-3β and the levels of the GSK-3β target β-catenin (32Woodgett J.R. Curr. Opin. Cell Biol. 2005; 17: 150-157Crossref PubMed Scopus (311) Google Scholar). To further confirm the role of Akt in cell survival, we have assessed the levels of Akt in cells transfected with Akt1 shRNA. We find that Akt levels are preserved in cells showing normal nuclear morphology and markedly reduced in cells showing condensed nuclei, indicating a strong correlation between cell death and low levels of Akt (Fig. 5D). These results suggest that Akt acting through GSK-3β signaling is necessary for the survival of PC12-D2R cells. Previously we have demonstrated that p53 and extracellular-regulated kinase (ERK) signaling plays an important role in mediating cell death and survival, respectively, in these cells (19Nair V.D. Apoptosis. 2006; 11: 955-966Crossref PubMed Scopus (72) Goog
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