Journal of Molecular Neuroscience

, Volume 59, Issue 4, pp 567–578 | Cite as

Involvement of PI3K/Akt/FoxO3a and PKA/CREB Signaling Pathways in the Protective Effect of Fluoxetine Against Corticosterone-Induced Cytotoxicity in PC12 Cells

  • Bingqing Zeng
  • Yiwen Li
  • Bo Niu
  • Xinyi Wang
  • Yufang Cheng
  • Zhongzhen Zhou
  • Tingting You
  • Yonggang Liu
  • Haitao WangEmail author
  • Jiangping XuEmail author


The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.


Fluoxetine Corticosterone PI3K/Akt/FoxO3a PKA/CREB Neuroprotection 



Protein kinase B


Brain-derived neurotrophic factor


Bcl-2-interacting mediator of cell death




cAMP-response element binding protein


Dimethyl sulfoxide


Forkhead box O3a




Methyl thiazolyl tetrazolium




Protein kinase A


p53 upregulated modulator of apoptosis


Ribosomal protein L19


Reverse transcription-polymerase chain reaction



This research was supported by National Natural Science Foundation of China (Nos. 81301099 and 81373384), Natural Science Foundation of Guangdong Province (No. S2013040014202), China Postdoctoral Science Foundation (No. 2013M542192), and National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (No. 2012ZX09J1211003C).

Compliance with Ethical Standards

Conflict of Interest Disclosures

The authors declare that they have no conflict of interest.


  1. Anacker C, Zunszain PA, Carvalho LA, Pariante CM (2011) The glucocorticoid receptor: pivot of depression and of antidepressant treatment? Psychoneuroendocrinology 36:415–425CrossRefPubMedPubMedCentralGoogle Scholar
  2. Beaulieu JM (2012) A role for Akt and glycogen synthase kinase-3 as integrators of dopamine and serotonin neurotransmission in mental health. J Psychiatry Neurosci 37:7–16CrossRefPubMedPubMedCentralGoogle Scholar
  3. Cecconi D, Mion S, Astner H, Domenici E, Righetti PG, Carboni L (2007) Proteomic analysis of rat cortical neurons after fluoxetine treatment. Brain Res 1135:41–51CrossRefPubMedGoogle Scholar
  4. Chen AC, Shirayama Y, Shin KH, Neve RL, Duman RS (2001) Expression of the cAMP response element binding protein (CREB) in hippocampus produces an antidepressant effect. Biol Psychiatry 49:753–762CrossRefPubMedGoogle Scholar
  5. Chiou SH, Chen SJ, Peng CH, Chang YL, Ku HH, Hsu WM, Ho LL, Lee CH (2006) Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell. Biochem Biophys Res Commun 343:391–400CrossRefPubMedGoogle Scholar
  6. Ciriaco M, Ventrice P, Russo G, Scicchitano M, Mazzitello G, Scicchitano F, Russo E (2013) Corticosteroid-related central nervous system side effects. J Pharmacol Pharmacother 4:S94–S98CrossRefPubMedPubMedCentralGoogle Scholar
  7. Djordjevic A, Djordjevic J, Elakovic I, Adzic M, Matic G, Radojcic MB (2012) Fluoxetine affects hippocampal plasticity, apoptosis and depressive-like behavior of chronically isolated rats. Prog Neuro-Psychopharmacol Biol Psychiatry 36:92–100CrossRefGoogle Scholar
  8. Freitas AE, Egea J, Buendia I, Navarro E, Rada P, Cuadrado A, Rodrigues AL, Lopez MG (2015) Agmatine induces Nrf2 and protects against corticosterone effects in hippocampal neuronal cell line. Mol Neurobiol 51:1504–1519CrossRefPubMedGoogle Scholar
  9. Geetha T, Rege SD, Mathews SE, Meakin SO, White MF, Babu JR (2013) Nerve growth factor receptor TrkA, a new receptor in insulin signaling pathway in PC12 cells. J Biol Chem 288:23807–23813CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gu W, Fukuda T, Isaji T, Hashimoto H, Wang Y, Gu J (2013) Alpha1,6-fucosylation regulates neurite formation via the activin/phospho-Smad2 pathway in PC12 cells: the implicated dual effects of Fut8 for TGF-beta/activin-mediated signaling. FASEB J 27:3947–3958CrossRefPubMedGoogle Scholar
  11. Hahn SJ, Choi JS, Rhie DJ, Oh CS, Jo YH, Kim MS (1999) Inhibition by fluoxetine of voltage-activated ion channels in rat PC12 cells. Eur J Pharmacol 367:113–118CrossRefPubMedGoogle Scholar
  12. Ishima T, Fujita Y, Hashimoto K (2014) Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells. Eur J Pharmacol 727:167–173CrossRefPubMedGoogle Scholar
  13. Kim HJ, Choi JS, Lee YM, Shim EY, Hong SH, Kim MJ, Min DS, Rhie DJ, Kim MS, Jo YH, Hahn SJ, Yoon SH (2005) Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores. Neuropharmacology 49:265–274CrossRefPubMedGoogle Scholar
  14. Kim HJ, Kim TH, Choi SJ, Hong YJ, Yang JS, Sung KW, Rhie DJ, Hahn SJ, Yoon SH (2013) Fluoxetine suppresses synaptically induced [Ca(2)(+)]i spikes and excitotoxicity in cultured rat hippocampal neurons. Brain Res 1490:23–34CrossRefPubMedGoogle Scholar
  15. King SC, Tiller AA, Chang AS, Lam DM (1992) Differential regulation of the imipramine-sensitive serotonin transporter by cAMP in human JAr choriocarcinoma cells, rat PC12 pheochromocytoma cells, and C33-14-B1 transgenic mouse fibroblast cells. Biochem Biophys Res Commun 183:487–491CrossRefPubMedGoogle Scholar
  16. Kolla N, Wei Z, Richardson JS, Li XM (2005) Amitriptyline and fluoxetine protect PC12 cells from cell death induced by hydrogen peroxide. J Psychiatry Neurosci 30:196–201PubMedPubMedCentralGoogle Scholar
  17. Krishnan V, Nestler EJ (2008) The molecular neurobiology of depression. Nature 455:894–902CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lecht S, Arienzakay H, Tabakman R, Jiang H (2007) Dexamethasone-induced down-regulation of nerve growth factor receptor p75NTR is mediated by glucocorticoid type II receptor in PC12 cell model. Open Pharmacol J 19:549–568Google Scholar
  19. Lee JY, Kang SR, Yune TY (2015) Fluoxetine prevents oligodendrocyte cell death by inhibiting microglia activation after spinal cord injury. J Neurotrauma 32:633–644CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lee JY, Lee HE, Kang SR, Choi HY, Ryu JH, Yune TY (2014) Fluoxetine inhibits transient global ischemia-induced hippocampal neuronal death and memory impairment by preventing blood-brain barrier disruption. Neuropharmacology 79:161–171CrossRefPubMedGoogle Scholar
  21. Li YF, Liu YQ, Huang WC, Luo ZP (2003) Cytoprotective effect is one of common action pathways for antidepressants. Acta Pharmacol Sin 24:996–1000PubMedGoogle Scholar
  22. Li YF, Liu YQ, Yang M, Wang HL, Huang WC, Zhao YM, Luo ZP (2004) The cytoprotective effect of inulin-type hexasaccharide extracted from Morinda officinalis on PC12 cells against the lesion induced by corticosterone. Life Sci 75:1531–1538CrossRefPubMedGoogle Scholar
  23. Li YF, Luo ZP (2002) Desipramine antagonized corticosterone-induced apoptosis in cultured PC12 cells. Acta Pharmacol Sin 23:311–314PubMedGoogle Scholar
  24. Liang B, Moussaif M, Kuan CJ, Gargus JJ, Sze JY (2006) Serotonin targets the DAF-16/FOXO signaling pathway to modulate stress responses. Cell Metab 4:429–440CrossRefPubMedGoogle Scholar
  25. Ma R, Hu J, Huang C, Wang M, Xiang J, Li G (2014) JAK2/STAT5/Bcl-xL signalling is essential for erythropoietin-mediated protection against apoptosis induced in PC12 cells by the amyloid beta-peptide Abeta25-35. Br J Pharmacol 171:3234–3245CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mao QQ, Huang Z, Ip SP, Xian YF, Che CT (2012) Protective effects of piperine against corticosterone-induced neurotoxicity in PC12 cells. Cell Mol Neurobiol 32:531–537CrossRefPubMedGoogle Scholar
  27. Mendez-David I, Tritschler L, Ali ZE, Damiens MH, Pallardy M, David DJ, Kerdine-Romer S, Gardier AM (2015) Nrf2-signaling and BDNF: a new target for the antidepressant-like activity of chronic fluoxetine treatment in a mouse model of anxiety/depression. Neurosci Lett 597:121–126CrossRefPubMedGoogle Scholar
  28. Morsink MC, Joels M, Sarabdjitsingh RA, Meijer OC, De Kloet ER, Datson NA (2006) The dynamic pattern of glucocorticoid receptor-mediated transcriptional responses in neuronal PC12 cells. J Neurochem 99:1282–1298CrossRefPubMedGoogle Scholar
  29. Pittenger C, Duman RS (2008) Stress, depression, and neuroplasticity: a convergence of mechanisms. Neuropsychopharmacology 33:88–109CrossRefPubMedGoogle Scholar
  30. Plattner F, Hayashi K, Hernandez A, Benavides DR, Tassin TC, Tan C, Day J, Fina MW, Yuen EY, Yan Z, Goldberg MS, Nairn AC, Greengard P, Nestler EJ, Taussig R, Nishi A, Houslay MD, Bibb JA (2015) The role of ventral striatal cAMP signaling in stress-induced behaviors. Nat Neurosci 18:1094–1100CrossRefPubMedPubMedCentralGoogle Scholar
  31. Polman JA, Welten JE, Bosch DS, de Jonge RT, Balog J, van der Maarel SM, de Kloet ER, Datson NA (2012) A genome-wide signature of glucocorticoid receptor binding in neuronal PC12 cells. BMC Neurosci 13:118CrossRefPubMedPubMedCentralGoogle Scholar
  32. Polter A, Yang S, Zmijewska AA, van Groen T, Paik JH, Depinho RA, Peng SL, Jope RS, Li X (2009) Forkhead box, class O transcription factors in brain: regulation and behavioral manifestation. Biol Psychiatry 65:150–159CrossRefPubMedGoogle Scholar
  33. Qi X, Lin W, Li J, Li H, Wang W, Wang D, Sun M (2008) Fluoxetine increases the activity of the ERK-CREB signal system and alleviates the depressive-like behavior in rats exposed to chronic forced swim stress. Neurobiol Dis 31:278–285CrossRefPubMedGoogle Scholar
  34. Sanphui P, Biswas SC (2013) FoxO3a is activated and executes neuron death via Bim in response to beta-amyloid. Cell Death Dis 4:e625CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sapolsky RM, Krey LC, McEwen BS (1984) Glucocorticoid-sensitive hippocampal neurons are involved in terminating the adrenocortical stress response. Proc Natl Acad Sci U S A 81:6174–6177CrossRefPubMedPubMedCentralGoogle Scholar
  36. Shin LM, Liberzon I (2010) The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology 35:169–191CrossRefPubMedGoogle Scholar
  37. Subramaniam S, Shahani N, Strelau J, Laliberte C, Brandt R, Kaplan D, Unsicker K (2005) Insulin-like growth factor 1 inhibits extracellular signal-regulated kinase to promote neuronal survival via the phosphatidylinositol 3-kinase/protein kinase A/c-Raf pathway. J Neurosci 25:2838–2852CrossRefPubMedGoogle Scholar
  38. Suzuki A, Fukushima H, Mukawa T, Toyoda H, Wu LJ, Zhao MG, Xu H, Shang Y, Endoh K, Iwamoto T, Mamiya N, Okano E, Hasegawa S, Mercaldo V, Zhang Y, Maeda R, Ohta M, Josselyn SA, Zhuo M, Kida S (2011) Upregulation of CREB-mediated transcription enhances both short- and long-term memory. J Neurosci 31:8786–8802CrossRefPubMedGoogle Scholar
  39. Tillinger A, Nostramo R, Kvetnansky R, Serova L, Sabban EL (2013) Stress-induced changes in gene expression of urocortin 2 and other CRH peptides in rat adrenal medulla: involvement of glucocorticoids. J Neurochem 125:185–192CrossRefPubMedGoogle Scholar
  40. Tiraboschi E, Tardito D, Kasahara J, Moraschi S, Pruneri P, Gennarelli M, Racagni G, Popoli M (2004) Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascades. Neuropsychopharmacology 29:1831–1840CrossRefPubMedGoogle Scholar
  41. Wakamatsu Y, Zhao X, Jin C, Day N, Shibahara M, Nomura N, Nakahara T, Murata T, Yokoyama KK (2001) Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERK-related signal cascade. Eur J Biochem 268:374–383CrossRefPubMedGoogle Scholar
  42. Wang J, Sun P, Bao Y, Liu J, An L (2011) Cytotoxicity of single-walled carbon nanotubes on PC12 cells. Toxicol in Vitro 25:242–250CrossRefPubMedGoogle Scholar
  43. Wang H, Duan X, Ren Y, Liu Y, Huang M, Liu P, Wang R, Gao G, Zhou L, Feng Z, Zheng W (2013a) FoxO3a negatively regulates nerve growth factor-induced neuronal differentiation through inhibiting the expression of neurochondrin in PC12 cells. Mol Neurobiol 47:24–36CrossRefPubMedGoogle Scholar
  44. Wang H, Zhou X, Huang J, Mu N, Guo Z, Wen Q, Wang R, Chen S, Feng ZP, Zheng W (2013b) The role of Akt/FoxO3a in the protective effect of venlafaxine against corticosterone-induced cell death in PC12 cells. Psychopharmacology 228:129–141CrossRefPubMedGoogle Scholar
  45. Wang H, Liao S, Geng R, Zheng Y, Liao R, Yan F, Thrimawithana T, Little PJ, Feng ZP, Lazarovici P, Zheng W (2015a) IGF-1 signaling via the PI3K/Akt pathway confers neuroprotection in human retinal pigment epithelial cells exposed to sodium nitroprusside insult. J Mol Neurosci 55:931–940CrossRefPubMedGoogle Scholar
  46. Wang H, Quirion R, Little PJ, Cheng Y, Feng ZP, Sun HS, Xu J, Zheng W (2015b) Forkhead box O transcription factors as possible mediators in the development of major depression. Neuropharmacology 99:527–537CrossRefPubMedGoogle Scholar
  47. Zheng WH, Kar S, Quirion R (2002) Insulin-like growth factor-1-induced phosphorylation of transcription factor FKHRL1 is mediated by phosphatidylinositol 3-kinase/Akt kinase and role of this pathway in insulin-like growth factor-1-induced survival of cultured hippocampal neurons. Mol Pharmacol 62:225–233CrossRefPubMedGoogle Scholar
  48. Zheng WH, Quirion R (2006) Insulin-like growth factor-1 (IGF-1) induces the activation/phosphorylation of Akt kinase and cAMP response element-binding protein (CREB) by activating different signaling pathways in PC12 cells. BMC Neurosci 7:51CrossRefPubMedPubMedCentralGoogle Scholar
  49. Zhou H, Li X, Gao M (2009) Curcumin protects PC12 cells from corticosterone-induced cytotoxicity: possible involvement of the ERK1/2 pathway. Basic Clin Pharmacol Toxicol 104:236–240CrossRefPubMedGoogle Scholar
  50. Zhou LJ, Zhu XZ (2000) Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide. J Pharmacol Exp Ther 293:982–988PubMedGoogle Scholar
  51. Zhou W, Chen L, Yang S, Li F, Li X (2012) Behavioral stress-induced activation of FoxO3a in the cerebral cortex of mice. Biol Psychiatry 71:583–592CrossRefPubMedGoogle Scholar
  52. Zhu MY, Ordway GA (1997) Down-regulation of norepinephrine transporters on PC12 cells by transporter inhibitors. J Neurochem 68:134–141CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Bingqing Zeng
    • 1
  • Yiwen Li
    • 1
  • Bo Niu
    • 1
  • Xinyi Wang
    • 1
  • Yufang Cheng
    • 1
  • Zhongzhen Zhou
    • 1
  • Tingting You
    • 1
  • Yonggang Liu
    • 2
  • Haitao Wang
    • 1
    Email author
  • Jiangping Xu
    • 1
    Email author
  1. 1.Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical SciencesSouthern Medical UniversityGuangzhouChina
  2. 2.Department of PharmacyGuangdong Provincial Corps Hospital of Chinese People’s Armed Police ForcesGuangzhouChina

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